Science topic

Transducers - Science topic

Transducers are any device or element which converts an input signal into an output signal of a different form. Examples include the microphone, phonographic pickup, loudspeaker, barometer, photoelectric cell, automobile horn, doorbell, and underwater sound transducer. (McGraw Hill Dictionary of Scientific and Technical Terms, 4th ed)
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Hello all, I am interested in purchasing a dox-inducible system that contains either GFP or RFP to transduce into cells and use for my animal studies. The cells will also contain luciferase as I will be detecting the growth of these cells in the animals using bioluminescence imaging. Will having GFP or RFP interfere with the bioluminescence signal and alter my results?
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Dear Brittany,
This literature provides a comparison between GFP luminescence and luciferase-luciferin imaging for your reference: PMID: 38925805.
GFP (Green Fluorescent Protein) has excitation/emission peaks at 397 nm and 509 nm, while RFP (Red Fluorescent Protein) exhibits peaks at 532 nm and 588 nm.
For luciferase-luciferin imaging experiments: The principle involves D-luciferin being catalyzed by luciferase to produce chemiluminescence at 560 nm. Importantly:
  • No excitation light within specific wavelength ranges is required
  • GFP and RFP will not generate fluorescent signals in this system、
Hope this helpful and feel free to reach out.
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Traditionally, we use silicon grease or gel, however, they are not available in my area.
I found silicon sealer.
Is it possible to be used as couplant for AE sensor?
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Vaseline makes a good couplant and is easily wiped off afterwards. Ideally in combination with magnetic holders.
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Hi All,
I am doing the lenti-transduction of viral constructs in Neural progenitor cells, I require at least 2 wells of one six well plate lentivirus prepared from 293T to infect the one 12 well plate of NPCs. The construct was tagged with GFP. It takes more time to get the enough ceIls for my experiment. I am using lenti-x-concentrator to prepare the lentivirus.
I require to do more conditions to transduce the NPCs. Is there any way that I can minimize the preparation time or/and any other way to prepare the lentivirus.
You Suggestions would be helpful
Thank you
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I don't know if this post is still relevant,
In order to minimize the preparation or to raise the lentivirus titer without adding supplemental cost or effort, I would suggest using our LentiBlast Premium Transduction enhancer.
LentiBlast Premium is a patented modified poloxamer that we developed a couple of years ago and that acts by simultaneously neutralizing electrostatic repulsions between membrane and viral particles and enhancing viral fusion with cell membrane. Due to a favorable “membrane permeable effect” limiting the transmembrane potential changes, it is non-toxic and totally compatible with cell viability.
It is a real asset to viral infection because it allows to increase the effectiveness of the viral vector, thus reducing the MOI, and consequently the immune response of the cell. Secondarily, it reduces the cost of experience. And thus I believe it would be ideal in your experiment.
LentiBlast Premium was demonstrated to be efficient of multiple cell types from Hematopoietic Stem Cells, to T lymphocytes and I am sure it would be worth give it a try for your experiment.
Some examples of publications with LentiBlast Premium:
  • HEK293: Brown AL. Sci Rep . 2021 Oct 25;11(1):21008.
  • Bone marrow derived macrophages: Fonseca GJ. Nat Commun. 2019 Jan 24;10(1):414.
  • Hematopoietic Stem Cells : Burton OT. Immunity. 2024 Jun 11:S1074-7613(24)00277-2.
  • HMEC : Nassour J. Nature . 2023 Feb;614(7949):767-773.
  • Myeloid progenitor : Verdys P. EMBO J. 2024 Jul 18. doi: 10.1038/s44318-024-00173-7.
  • T lymphocytes CD4+ : Claireaux M. Nature Commun 2022 Jan 26;13(1):521.
  • THP-1 : Chirayath TW. Nat Commun. 2024 Sep 18;15(1):8179.
Would you like to give LentiBlast a try, please do not hesitate to contact me via ResearchGate or directly at: tech@ozbiosciences.com
best regards,
Cedric
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I am not able to transduce C2C12 cells with lentivirus-GFP. I seeded 75,000 cells in a 6 well plate and I transduced after 24hrs. But they become too crowded at 48hrs and did not show GFP+ cells
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The optimal cell density for transducing C2C12 cells with lentivirus is typically when the cells are at a subconfluent state, ideally less than 50% confluence. This allows for efficient viral entry and reduces cell-to-cell contact, which can hinder viral infection. For a 6-well plate, seeding around 30,000 to 50,000 cells per well 24 hours before transduction can help maintain an optimal density at the time of transduction (Tapia O., Methods Mol Biol. 2016).
Now regarding Lentiviral transduction and selection process with antibiotics, I would recommend two options:
1. Choose the correct antibiotic (ab) concentration: prepare a cell plate (24-well plate) and add increasing doses of ab. 48H to 72H after observe what minimal dose is lethal for your cells and work with that. Depending on the cell line and culture condition it may vary a lot.
2. Try our LentiBlast Premium Transduction enhancer to increase viral infection.
LentiBlast Premium is a patented modified poloxamer that we developed a couple of years ago and that acts by simultaneously neutralizing electrostatic repulsions between membrane and viral particles and enhancing viral fusion with cell membrane. Due to a favorable “membrane permeable effect” limiting the transmembrane potential changes, it is non-toxic and totally compatible with cell viability.
It is a real asset to viral infection because it allows to increase the effectiveness of the viral vector, thus reducing the MOI, and consequently the immune response of the cell. Secondarily, it reduces the cost of experience. And thus I believe it would be ideal in your experiment.
LentiBlast Premium was demonstrated to be efficient of multiple cell types from Hematopoietic Stem Cells, to T lymphocytes and I am sure it would be worth give it a try for your experiment.
Some examples of publications with LentiBlast Premium:
  • HEK293: Brown AL. Sci Rep . 2021 Oct 25;11(1):21008
  • Bone marrow derived macrophages: Fonseca GJ. Nat Commun. 2019 Jan 24;10(1):414.
  • Hematopoietic Stem Cells : Burton OT. Immunity. 2024 Jun 11:S1074-7613(24)00277-2.
  • HMEC : Nassour J. Nature . 2023 Feb;614(7949):767-773.
  • Myeloid progenitor : Verdys P. EMBO J. 2024 Jul 18. doi: 10.1038/s44318-024-00173-7.
  • T lymphocytes CD4+ : Claireaux M. Nature Commun 2022 Jan 26;13(1):521.
  • THP-1 : Chirayath TW. Nat Commun. 2024 Sep 18;15(1):8179.
Would you like to give LentiBlast a try, please do not hesitate to contact me via ResearchGate or directly at: tech@ozbiosciences.com
best regards,
Cedric
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We attempted to transduce NK-92 cells using lentivirus. The virus was produced in HEK293T cells, yielding a biological titer of 3×10⁶ particles per mL. We concentrated the virus using two different methods: PEG precipitation and Amicon (100 kDa) filtration, maintaining similar final concentrations.
For transduction, we used:
  • Filter-concentrated virus at MOI 10, 20, 30, 40, and 50 (no positive results).
  • PEG-concentrated virus at MOI 10 and 15 (11% positivity at MOI 10).
However, with PEG-concentrated virus, we observed a 50% cell loss due to cell death. Additionally, after changing the medium, all NK-92 cells died within 24 hours.
Has anyone encountered similar issues? What strategies could improve transduction efficiency while minimizing toxicity? Any recommendations would be highly appreciated!
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Dear Masoud,
It is true that NK cells, primary as well as cell lines, are difficult to genetically modify, even with a lentiviral vector. One problem may be the MOI to use, and especially if the viral particles are “only” concentrated and not purified. A high dose of virus can induce a massive non expected innate immune response from the target cells that will lower the overall efficiency.
This is why it may be interesting to lower the MOI while increasing the efficiency, and to this end, I would like to propose you to try our LentiBlast Premium.
LentiBlast Premium is a patented modified poloxamer that we developed a couple of years ago and that acts by simultaneously neutralizing electrostatic repulsions between membrane and viral particles and enhancing viral fusion with cell membrane. Due to a favorable “membrane permeable effect” limiting the transmembrane potential changes, it is non-toxic and totally compatible with cell viability.
It is a real asset to viral infection because it allows to increase the effectiveness of the viral vector, thus reducing the MOI, and consequently the immune response of the cell. Secondarily, it reduces the cost of experience.
LentiBlast Premium was demonstrated to be efficient of multiple cell types from Hematopoietic Stem Cells, to T lymphocytes and I am sure it would be worth give it a try for NK cell type.
Some examples of publications with LentiBlast Premium:
  • Bone marrow derived macrophages: Fonseca GJ. Nat Commun. 2019 Jan 24;10(1):414.
  • Hematopoietic Stem Cells: Burton OT. Immunity. 2024 Jun 11:S1074-7613(24)00277-2.
  • HMEC: Nassour J. Nature . 2023 Feb;614(7949):767-773.
  • Myeloid progenitor: Verdys P. EMBO J. 2024 Jul 18. doi: 10.1038/s44318-024-00173-7.
  • T lymphocytes CD4+: Claireaux M. Nature Commun 2022 Jan 26;13(1):521.
  • THP-1: Chirayath TW. Nat Commun. 2024 Sep 18;15(1):8179.
Would you like to give LentiBlast a try, please do not hesitate to contact me via ResearchGateor directly at: tech@ozbiosciences.com
best regards,
Cedric
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I transduced NK92 cells and primary NK cells with lentivirus. The transduction efficiency was acceptable initially. However, NK92 cells died three days post-transduction. As for primary NK cells, although they remained viable, the transduction efficiency dropped to zero by day 6 post transduction.
I expected some reduction in efficiency over time, but not a complete loss. I even sorted the positive cells, but this did not improve the outcome.
Has anyone encountered this issue before or have any suggestions on how to prevent efficiency loss and cell death?
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Dear Yulin Wang,
It is true that NK cells, primary as well as cell lines, are difficult to genetically modify, even with a lentiviral vector. To prevent efficiency loss and may be cell death (that could be a consequence of the infection process – maybe due to a high MOI), I would like to propose you to try our LentiBlast Premium.
LentiBlast Premium is a patented modified poloxamer that we developed a couple of years ago and that acts by simultaneously neutralizing electrostatic repulsions between membrane and viral particles and enhancing viral fusion with cell membrane. Due to a favorable “membrane permeable effect” limiting the transmembrane potential changes, it is non-toxic and totally compatible with cell viability.
It is a real asset to viral infection because it allows to increase the effectiveness of the viral vector, thus reducing the MOI, and consequently the immune response of the cell. Secondarily, it reduces the cost of experience.
LentiBlast Premium was demonstrated to be efficient of multiple cell types from Hematopoietic Stem Cells, to T lymphocytes and I am sure it would be worth give it a try for NK cell type.
Some examples of publications with LentiBlast Premium:
  • Bone marrow derived macrophages: Fonseca GJ. Nat Commun. 2019 Jan 24;10(1):414.
  • Hematopoietic Stem Cells: Burton OT. Immunity. 2024 Jun 11:S1074-7613(24)00277-2.
  • HMEC: Nassour J. Nature . 2023 Feb;614(7949):767-773.
  • Myeloid progenitor: Verdys P. EMBO J. 2024 Jul 18. doi: 10.1038/s44318-024-00173-7.
  • T lymphocytes CD4+: Claireaux M. Nature Commun 2022 Jan 26;13(1):521.
  • THP-1: Chirayath TW. Nat Commun. 2024 Sep 18;15(1):8179.
Would you like to give LentiBlast a try, please do not hesitate to contact me via ResearchGateor directly at: tech@ozbiosciences.com
best regards,
Cedric
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Good day!
I currently am doing research on the effects of ultrasonication on electroplating. However, I am looking for alternatives to the costs of purchasing a probe sonicator for the experimental setup. Based on the prices I've seen online, creating your own ultrasonic bath setup is way cheaper than buying the probe sonicator. However, I have yet to find a good resource for the creation of the system.
I hope someone who's more familiar can enlighten me on my working plan.
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Have you tried using the drive circuitry and the transducer from an ultrasonic humidifier?
Regards,
Tom Cuff
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I am looking for an ultrasonic transducer with a frequency range of 40-50 kHz for measuring wood thickness up to 200 mm (not for distance measurement). I need the emitter to send the sound and the receiver to return the signal when in contact with the object. Please, do you have any suggestions for a model?
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If you're looking for a contact transducer coupled to your specimens using a suitable couplant, an X1021 transducer from Evident (former Olympus, Parametrics) might be fitting (link below). I suppose you could also use a horn transducer without a couplant but I can't find a supplier by a quick web search.
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do you know if it’s possible to generate and detect ultrasound with electromagnetic transducers on lead (Pb)?
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Hello.
Electromagnetic acoustic transducers (EMATs) work by generating ultrasonic waves in a test object using electromagnetic induction. Here's how EMATs work on lead or other electrically conductive objects:
  1. 1. Create eddy currentsAn EMAT consists of a magnet and an electrical coil. When an alternating current passes through the coil, it creates eddy currents in the surface layer of the metal.
  2. 2. Apply a static magnetic fieldThe static magnetic field exerts a force on the eddy currents.
  3. 3. Oscillate particlesThe oscillating force causes the particles of the component to oscillate, generating an acoustic wave.
  4. 4. Transfer disturbance to the materialThe disturbance is transferred to the lattice of the material, producing an elastic wave.
EMATs can generate various types of waves, including shear waves with horizontal polarization (SH waves). They are the only practical way to generate these waves. EMATs are advantageous over piezoelectric transducers because they don't load the surface of the object being monitored. This eliminates problems with reverberation processes.
Thanks,
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Hi everyone! I work with NK-92 cell line (ATCC) and am trying to transduce them with a CAR. After several attempts of transduction, I’ve achieved a place where I feel hopeless. I’ve tried to alter many different variables in the protocol, since at the virus production stage - using HEK 293-T cells and lipofectamine 3000 transfection protocol - till enhancements in the transduction protocol. I concentrated the virus, used Vectofusin and spinoculation to improve transduction, tested my protocols on other cell types and they’re working. I am now producing the viral particles with a vector for the envelope that is supposed to be more effective on NK cells that the commonly used VSVG, BaEV-LV, and I‘ve also tried the same protocol that the authors present (Bari R et al, 2023; doi: 10.3389/fimmu.2019.02001), but apart from one attempt (in which the cells stopped proliferating after transduction and slowly died, and which I could not replicate, even doing exactly the same steps), I still didn’t get an efficiency rate greater than 3%. I don’t know if there’s a tip or any determinant step in the protocol that I’m missing, but any kind of help would be very welcome!
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As attached.
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Necessary information-
1. Target cell line: mutuDC/ DC1940 (mouse CD8a+ immortalized cells). Passage is over 25 (early passage not available). Cells are cultured in IMDM media (10% FBS, PenStrep, Sodium bicarbonate, HEPES and 2-mercaptoethanol)
NOTE: Highly sensitive cell line. Needed to be handled very carefully. siRNA mediated transduction is not feasible.
2. Plasmid: pLKO.1 plasmid with shRNA against a target long non-coding RNA (not commercially available so had to clone it myself).
3. Virus particles generated using CalPhos kit in HEK293T cells (cultured in DMEM). Virus collected at 24hrs, centrifuged and filtered using 0.45 micron syringe filter, aliquoted and stored at -80C.
4. Transduction: Cells are seeded at 0.15x106/ well in 12-well plates. After 12-14hr, each viral supernatant were added as follows: 50ul > 100ul > 150ul > 200ul. Total media in per well was 300ul. After 10hr of incubation, viral media was changed and fresh media was added. Media was changed after every 24hr as there was a lot of cell death (no extra supplement of FBS or other reagents were used in the media). After 72hr of transduction, Puromycin was added at the conc. of 0.5ug/ml (based on kinetics done earlier) in the same well where the mutuDCs were transduced (cells were not transferred to fresh plate as they will go under stress).
PROBLEM: After 4-5days of Puromycin selection, the (supposedly) transduced cells are proliferating well in 12-well plate. After each well was about 60%-70% confluent, the cells were dissociated (using non-enzymatic dissociation media; contains PBS and EDTA) and transferred into 6-well plate (from one well of 12-well plate to one well of 6-well plate). Within 14hr-16hr the cells started going into stress (swollen, serrated cell membranes, etc.) and after 24-30hr they started dying. I even tried transferring cells from one well of 12-well plate to two wells of 12-well plate (thinking maybe surface area could be the issue), but still faced the same problem. Now the cells are not under Puromycin selection as it might further add to the stress.
Kindly provide any and all suggestions as per your knowledge to help me out of this issue. Thank you in advance.
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First of all, perform the infection experiment using freshly prepared virus containing medium without freezing the virus.
Freezing lentivirus at -80°C will reduce titers by a x10 or more.
Second, why is the virus-containing media collected and centrifuged before filtering? Because we are only removing cell debris by filtering, pass the virus-containing medium through the filter without centrifuging.
In gene overexpression experiments using lentivirus, freshly collected virus-containing medium will be diluted 1/4~1/8 for infection, but in KD experiments using pLKO.1, we use virus-containing medium with 1/2 dilution or undiluted for infection. For this protocol, HEK293T must me cultured in the same culture medium as target cells.
If you want to freeze lentiviruses, you can mix PEG-8000 and precipitate the lentiviruses with PEG as a cryoprotectant, preventing a significant decrease in titer.
Virus Precipitation Solution (PEG-it) is commercially available, but you can also make your own as described in the following paper.
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I transduced my cells with 2ml of the viral supernatant and 4ug/ml polybrene. After transduction, 16 hours, I replaced the media with normal media. I started antibiotic selection with 6ug/ml blasticidin 48 hours after transduction. During selection there was a lot of cell death, although compared to my positive control some cells remained in my T75s. I selected for just one week/ when all the cells in my positive control flask were dead. However these cells are in these little cell islands, and there are very few cells. They appear to be slow growing and I am not confident that my flask will become confluent. Can someone tell me what I could have done wrong?
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the cells were likely stressed. be as gentle as possible - if you can use PBS + EDTA to release rather than trypsin I would try that (called enzyme-free cell dissociation buffer or versene or you can look up a recipe and make it, works on cells that are not firmly adhered). pipet very gently.
you could try inoculating cells in a well-plate like 24-well or 96-well, then growing out. or you can put colony isolation rings over the small groups of cells but that would work on a plate, not a flask.
also it depends on the cell type and the dish, perhaps adding an additional coating might help them adhere - the type you use would depend on what is accepted to use for the cell type. poly-d-lysine coating is usually accepted for most cells. otherwise collagen or fibrinogen could work. however, do NOT do this if it is not well-published for your cell type as it would be a new protocol and reviewers will come after you when a paper is submitted.
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Generally, the BVD model is used to investigate the electrical characteristics of the emitting piezoelectric ultrasonic transducers. When piezoelectric ultrasonic transducers are used to receive acoustic signals, what equivalent method or equivalent circuit is used to characterize them?
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As usual, it depends! (The universal answer to any questions worth asking.) For higher frequencies, I've found the KLM model -- Electron Letters, 6, no. 13, 398-399 easiest with thin layers. If you have it, George Kino's "Acoustic Waves", Prentice Hall, 1987, Section 1.4, discusses using the various models and when they might be used quite nicely....
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Hello,
I transduced gastric cancer cell lines with Tet-inducible plasmid that have my gene of interest. Now, I need to find the concentration and duration of tetracycline treatment that can activate my gene of interest. What range of concentration and duration do you suggest? (I have three kinds of plasmids: overexpression, knockdown and control (backbone) of my gene of interest).
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Hi Fariba,
Maybe will help you,
Kind regards,
Lara
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Hello,
I will appreciate if anyone can help with a protocol to either transduce or transfect IPSc as I am planning to do this for the first time
Thank you
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Charlie Amoia Thank you for the explanation and your time
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Hey, I have been trying to transduce cardiac fibroblasts with lentiviruses. I transduced them using different concentrations of non-concentrated as well concentrated viruses. I went for as high as 100uL of the concentrated lentiviruses. But when I start selection of the transduced cells with puromycin all the cells are dead. And also the cells do not express the reporter gene (mCherry). Seeking advice on identifying and resolving the underlying issue causing these outcomes.
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If you explain the time-course experimental procedures, we might be able to give you some advice.
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I would appreciate help here in avoiding re-inventing the wheel.
Has anyone used AAV to transduce THP-1 in suspension culture?
I do not want to do this in activated adherent (macrophage-like) THP-1, but rather in suspension.
If so I would love a protocol as far as multiplicity of infection, cell density, media switching/media starvation, timing, and any useful notes.
Thanks everyone,
Neal
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Hi Neal,
We have performed transfection of THP-1 with AAV2 and AAV6 with MOI ranging from 1k to 200k. Our protocol so far: Plate 100k cells per well in an untreated round bottom 96 well plate. Spin down and aspirate all but 20uL of media. Add AAV at desired MOI and spinoculate for 2 hours at 32C. Add 100 uL of media and incubate overnight. We observed transfection the following day via flow.
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Hi there, I am attempting to transduce cells with the TLCV2 plasmid containing our sgRNA of interest. My transfection of the HEK 293T cells seems to yield positive results as the EGFP is clearly expressed by my cells under the microscope. I harvest the media at 48 hours and then allow my target cells to sit for 24 hours without disturbing them. I then selected them with puromycin for 3 days at 4ug/ml which is well above what is needed to kill the controls but I have been concerned about residual non transduced cells. After selection though, when I go to image my cells they do not express the EGFP gene. This has been shown to be previously successful by members in our lab so I am at a loss for the cause. I take the media directly from the 293T cells, spin it, and filter with .44 filter. I do not concentrate the virus at all. I have yet to run a western blot to see if my GOI is knockdown as I do not want to waste a large chunk of time if I can know my transduction did not work from the EGFP.
Does anyone have experience with this issue? If so how did you resolve it? Any help is appreciated thank you.
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A quick glance at the TLCV2 vector from addgene...it's an all-in-one tet-inducible....have you tried adding some doxy to your transduced cells? According to the map, the backbone expresses Cas9 2A GFP...so your GFP will be under doxy induction as well.
As these promoters are sometimes leaky, you are likely seeing GFP in your packaging cells because of the quantity of DNA you transfected (many per cell). Viral transductions are much less, and therefore, you may not be able to visualize the small leakiness.
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My transduced cell can resist Puromycin and 99% express GFP, but it cannot show me the difference in the knockdown. What could be the possible reason?
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Did you try ran a PCR to see if the gene that should be knockdown presented no band?
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Does anyone have a working protocol or suggestion for CRISPR knockout gene in THP-1 cell. I worked for more than half year to knock CFB gene in THP-1 cells. When I transduced the CAS9 into THP-1 at the beginning, after I applied blasticidin 99% cells died, very small portion of cells didn't proliferate, and finally all cells died. I got CAS9 expressing THP-1 cell at the third try. But I still cannot get single cell clone by limited dilution. Because the single cell didn't proliferate, all the cells died later.
It was even exceedingly difficult to transduce the guide RNA into the CAS9 expressing THP-1 cells. I tried twice, all the cells died after apply the selection antibiotics hygromycin. The transduced cells even died faster than the untransduced cells.
Any suggest and help are welcome.
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Hi Zeyu;
Did you solve this problem?
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I am performing PCR as a QC test to look for a transcription gene that should be negative after a CAR T therapy process. As we are comparing against a CAR transduced patient's cell, we require a used transduced ATCC cells. However, the ATCC cells have a low transfection titer, which makes the PCR band faint and when kept for long, it becomes fainter and fainter.
I was thinking of using another different grade of cells such as transduced research grade cells as it was observed that the bands tend to be much brighter than the ATCC grade cells.
Is it possible to use transduced research grade cells instead of ATCC grade?
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Using a different grade of cells for your positive control could potentially introduce variability or inconsistency into your experiment. It's generally recommended to use the same grade or quality of cells for both experimental and control groups to ensure reliable and accurate results. However, if you have valid reasons for using a different grade of cells for your positive control, it's essential to thoroughly validate and justify this decision to ensure the integrity of your experimental findings.
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We have constructed lentivirus transduced U251 cell lines to express (1) EGFRvIII (its amino acid sequence is MRPSGTAGAAFLALLAALCPASRALEEKKGNYVVTDHG..., which is identical to the sequence described in https://www.addgene.org/20737/)-mCherry or (2) EGFRvIII_G38C(MRPSGTAGAAFLALLAALCPASRALEEKKGNYVVTDHC...)-mCherry.
Both types of cells did not yield positive results by using antibodies against LEEKKGNYVVTDHC (https://www.novusbio.com/products/egfr-antibody-dh83_nbp2-50599af647).
The (2) EGFRvIII_G38C-U251 cells have not been tested by 2-step staining.
We are curious about this question. Have you met problems when dealing with EGFRvIII?
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Antibodies against EGFRvIII, a variant of the epidermal growth factor receptor (EGFR) that is commonly found in certain types of cancer, may not work effectively for several reasons such as Tumor Heterogeneity, Immune Evasion Mechanisms, Resistance Mechanisms, Blood-Brain Barrier, Inadequate Targeting.
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I wonder if there is a clear version of "Theoretical analysis of relations between directivity of SAW transducer and its dispersion curves"?
DOI: 10.1109/ULTSYM.1989.66961
In the current version on IEEE, figures and equations are almost indistinguishable.
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I do not think, since the directivity (if you do not mean also reflectance) depends (as well) on the aperture of the SAW structure relative to the signal's wavelength.
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what are the possible reason(s) that make AsPC-1, 293T and MCF-7 cell lines failed to be transfect/transduced?. Please help me with possible hints?
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You may consider the following reasons.
1. The choice of which cell type to use for transfection is a critical factor that is often overlooked. Since each cell type is likely to respond differently to a given transfection reagent, choosing an appropriate cell type is necessary to maximize results. For instance, some cell types like MCF7 or HepG2, prefer to grow in clumps or clusters which is not ideal for transfection because minimal membrane surface is exposed which compromises uptake. Blood or immune cells that lack the proper endocytic machinery can minimize uptake and there are the macrophages that have an evolved uptake mechanism, but quickly breakdown and destroy endosomal contents. So, select the right cell line for transfection.
2. Make sure that the cells are not 100% confluent (should be about 60-80% confluent) or in stationary phase at the time of transfection because actively dividing cells take up foreign nucleic acid better than quiescent cells.
3. Most cell types should be used between 4 and 25 passages for optimal transfection.
4. There could be a possibility that the DNA concentration may be too low, or DNA may be degraded. In such a case, you may increase the ratio of DNA :transfection reagent, and you may confirm DNA integrity by A260/A280 spectrophotometer reading (should be at least 1.7).
5. You may use serum-free media for DNA dilution because some serum proteins may interfere with complex formation. You may also increase complexing reaction incubation time.
6. Test the culture for contamination.
7. Do not use antibiotics at the time of transfection because cationic lipid reagents increase cell permeability. As a result they may also increase the amount of antibiotics delivered into the cells, causing cytotoxicity and low transfection efficiency.
8. Cells could suffer mechanical damage during the experimental steps. So, do not vortex or spin cells for extended period.
9. Some transfection reagents could be compromised due to improper storage.
Best.
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I have knocked down my gene of interest by lentiviral transduction. I revived the vials and continue growing cells for 72 h without any selection. After confluency, I passaged the cells later 24 hours I have used puromycin 1ug/ml, 3ug/ml, 5ug/ml for selection of transduced cells. After 2 days, most of my virus transduced cells die at all the puro conc. The viral transduction itself doesn't seem to be toxic as cells were growing up until selection. Any suggestions would be greatly appreciated!
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What kind of cells? What is a vector design? How are You sure if the lentivirus entered the cells and integrated?
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I have tried to read about this but I don't find definitive answers.
I am trying to lentivirally transduce a mouse cell line with a few different plasmids (each encoding a different antibiotic resistance gene, namely puromycin, hygromycin and G418). After successfully transducing the cells a first time (with the puromycin resistance-containing plasmid), it now looks like when I transduce the cells with the hygromycin resistance-containing plasmid they don't die at the hygromycin concentration established during the antibiotic titration.
Has anyone experienced this?
I found this paper, but it doesn't seem to answer my question fully: https://www.sciencedirect.com/science/article/abs/pii/S0003269709008434
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Hi Carles Puig,
I haven't faced a problem like that. However I can share you some considerations:
1) Have you considered non-simultaneos transduction of your plasmids? For instance, first you transduce a plasmid, then you perform a selection of 2-3 days, then another plasmid with its subsequent selection and so on...
2) In our lab we had a couple of problems with G418, it was because of the analogy with neomycine and hygromicine. Maybe you can check thoroughly if the gene resistance can show cross-reaction, and if so, change one of the antibiotics
What type of cells are you transducing/transfecting?
I hope it helps
Best regards,
Francesc
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I am need some help with T-cell expansion. I have isolated PBMCs from healthy human blood and activated T-cells. After transducing T-cells with lentivirus I am facing hard time expanding Transduced T-cells. They grow really slow. Can anyone help me with this? I activate T-cells after transduction every 2nd day.
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designed to promote their growth and proliferation while maintaining their functionality. This process is crucial in research and therapeutic applications, such as CAR-T cell therapies. Here's a general protocol to guide you through the expansion of transduced T cells:
1. Preparation Before Transduction
  • Activation: Start by activating the T cells, as activated cells transduce more efficiently with lentivirus. Use activation beads coated with anti-CD3 and anti-CD28 antibodies, or other activation methods suitable for your T cell subtype. Activation typically occurs 1-2 days before transduction.
  • Culture Conditions: Keep the cells in a T cell growth medium supplemented with cytokines, such as Interleukin-2 (IL-2), which is critical for T cell proliferation. The concentration of IL-2 can vary, but 50-300 IU/mL is commonly used.
2. Transduction Process
  • After activation, transduce the T cells with lentivirus. The multiplicity of infection (MOI; ratio of viral particles to target cells) is crucial and should be optimized based on your vector and goals. MOIs of 5-10 are common, but this can vary widely.
  • Enhance transduction efficiency by centrifuging the plates (spinoculation) at 800-1000 g for 1-2 hours at 32°C and then incubating them at 37°C in a CO2 incubator.
  • After 12-24 hours, replace the medium with fresh T cell growth medium containing IL-2 to remove viral particles and support cell growth.
3. Post-Transduction Expansion
  • Medium Refreshment: Regularly refresh the culture medium every 2-3 days, adjusting the concentration of IL-2 based on cell proliferation rates and condition. Watch for cell density; too high or too low can affect growth and functionality.
  • Cell Density: Maintain an optimal cell density, typically between 0.5 to 1 million cells/mL, by adjusting the culture volume or splitting the cells into new flasks as necessary.
  • Monitoring: Keep a close eye on cell viability and growth. Use flow cytometry to assess the percentage of cells expressing the transgene, which can also help in monitoring the success of the transduction.
4. Expansion Phase
  • As the T cells proliferate, gradually scale up the culture volume or transfer the cells to larger flasks/bioreactors. The expansion strategy will depend on your specific application and the required cell number.
  • Continue supplementing the culture with IL-2 and monitoring cell health, density, and transgene expression.
  • For large-scale expansions, systems like gas-permeable culture bags or bioreactors can be used to manage larger volumes of cells efficiently.
5. Harvesting
  • Once the desired expansion level is reached, cells can be harvested for further use. Centrifuge the cells, and resuspend them in the appropriate buffer or medium for your application.
  • Perform a final assessment of cell count, viability, and functionality (e.g., cytotoxicity assays for CAR-T cells) before use.
6. Cryopreservation (Optional)
  • If you're not using the expanded T cells immediately, you can cryopreserve them for long-term storage. Use a cryopreservation medium with a cryoprotectant like DMSO and store the cells in liquid nitrogen.
Key Considerations
  • Safety: Work in a BSL-2 (or higher) facility when handling lentivirus and follow all institutional biosafety guidelines.
  • Cytokine Support: The choice and concentration of cytokines can significantly affect the expansion and functionality of T cells. Some protocols may also include additional cytokines like IL-7 and IL-15.
  • Optimization: Optimize the activation, transduction, and expansion conditions based on your specific T cell subtype and application.
Following these steps can help achieve successful expansion of transduced T cells, maintaining their viability and functionality for research or therapeutic use.
l This list of protocols might help us better address the issue.
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For several months now, I've been attempting to generate and infect my cells with lentiviruses, but unfortunately, I have encountered persistent challenges. The specific cells I aim to transduce are human fetal fibroblasts (HFF), and the plasmid I'm using is "dCas9-5xGCN4-P2A-BFP" with Addgene number 184438. Interestingly, this plasmid has demonstrated success in transducing other cell lines for different research groups. However, after thorough investigation, I have ruled out the cells themselves, our protocol, and personal error as potential causes for the unsuccessful transduction.
It's worth noting that I have successfully created lentiviruses and transduced HFF cells with high efficiency using other plasmids. Despite my efforts, the lentiviral concentration and the spinoculation method for transduction have not yielded positive results.
If you have any insights or advice to offer regarding this issue, I would greatly appreciate your input.
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Ok thanks for the information. I suspect, given the large size of dCas9-5xGCN4-P2A-BFP, that the RNA is not being efficiently packaged into the virus. The larger the RNA, the less efficient packaging is, resulting in virus particles with no nucleic acid in them. I have experienced this previously and the amount of lentivirus produced/titer was extremely low resulting in unsuccessful transduction of the target cells.
You may want to contact the research groups that had success in transducing this plasmid and find out how they produced their lentivirus.
Good luck!
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Hello, I want to tranduce the above cell lines using lentivirus. I will appreciate help from anyone that have experience transducing any of these cell lines especially the best MOI and cell number used. Thank you
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Yes, works great!
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If I want to transduce cells with two lentiviral vectors, do I perform the transduction one after the other? Or do I collect the lentivirus from the different HEK plates and co-transduce my target cells?
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Isn't it better to perform a transduction with a polycistronic vector? Or do you want to test different MOIs?
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No longer available
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Thanks again Qamar Ul Islam Just out of curiosity: are you using ChatGPT to generate these answers?
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Hello, I'm trying to knock down and re-express (both through adenovirus) a protein of interest. My knock down using shRNA targeted to 3' UTR is pretty efficient, but when I try to re-express the protein after knock down, I don't get a full rescue. Specifically, some cells appear to re-express the protein well, but others not so much and still others not at all.
I've attached a couple IF images to show you what I get. Red is antibody against protein of interest and green is the GFP signal from the exogenous protein. As you can see, when I transduce control cells (not shRNA knock down) with the rescue construct, most of the cells appear to get the GFP. When I transduce with shRNA adenovirus prior to rescue, I get a mediocre rescue.
If relevant, this is the rough protocol I follow:
D1: Transduce cells with shRNA adenovirus
D2 morning: change media to complete media
D2 afternoon/evening: wash cells with 1X DPBS, transduce with rescue adenovirus
D3: change media to complete media
D4: harvest/fix/etc
Also, I'm a graduate student still learning so I'd appreciate it a lot if you can explain things in a way that isn't too technical. Thanks for the help in advance.
Edit: Researching further, it became apparent that my protein which forms filament structures cannot form those structures with only the GFP-tagged version. It requires some wild type to be present in order to build upon it. I guess some of my cells got such a good knockdown that it couldn't be rescued by the GFP-tagged construct. I'll leave this question up so that anyone with similar issues can see if this might be the case for them.
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What about reinfection a second time? Have you tried? Pardon the comment in 2024
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Hi All,
I need an opinion about an experiment I am planning to do.
I am planning to do a genome wide CRISPR/Cas9 screening to identify genes responsible for drug resistance. I have purchased the Brunello lentiviral library and am planning on transducing cells with the virus particles. My question has to do with the screening part: according to the library preparation protocol from Broad Institute, the PCR is done with 96 different P7 primers in a 96-well format (protocol in link: https://media.addgene.org/cms/filer_public/61/16/611619f4-0926-4a07-b5c7-e286a8ecf7f5/broadgpp-sequencing-protocol.pdf) . I have come across other protocols, published both before and after my reference paper, which have used 8-10 P5 and 8-10 P7 primers (instead of the 96 P7 primers used here), as is outlined here, for example : https://star-protocols.cell.com/protocols/524.
Using 96 primers will increase the experiment cost, as these are 91-mer primers. Primers itself will cost $1700, whereas using 10 primers will cost $170.
Does anybody have any inputs as to what is to be kept in mind when I am deciding which option I should go for as far as coverage is concerned?
Thanks in advance!
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Well, the primer cost is low compared to all the other associated costs such as salaries and time. I’d use the protocol that seems most likely to get you high quality results.
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I had a miscommunication with a coworker, and as a result I put unfiltered virus harvested from 293T Phoenix media on to my primary fibroblasts. The transduction worked well, but now I have a couple contaminant Phoenix cells on my culture dishes.
I have tried several days of aggressive washing, since the Phoenix cells are easily detached, but that has been unsuccessful so far at removing all of them. The primary cells I have successfully transduced are very precious, so any other ideas at removing the Phoenix cells are appreciated.
As a last resort, I can flow sort the cells into single wells and grow them out from there. Hoping to avoid it for now as it is incredibly time consuming and also stresses out the cells.
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HEK derived cells like Phoenix do not like cooler cultivation temperatures unlike fibroblasts (https://pubmed.ncbi.nlm.nih.gov/7276615/).
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I'm looking for some cell lines that AAV9 will transduce efficiently at 10^6 MOI besides CHO-Lec2. I've already tested CHO-Lec2 and found decent transduction levels (~20%). But now, I would like to gather additional data in other cell lines. Any suggestions? I've heard maybe U87 cells are good for AAV9? Let me know your thoughts, thank you!
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Adeno-associated vectors (AAV) has a remarkable ability to transduce astrocytes, neurons, and endothelial cells in the brain and spinal cord. You could try DRG-derived cell lines.
You may want to refer to the articles attached below.
You could also try Neuro-2a (N2a) cells, a mouse brain neuroblastoma cell line.
Please see article attached below.
U87 cells are also good for AAV9.
Best.
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What is the best cell line for Cardiomyocytes for in vitro functional assay. I need a robust cell line easy to transduce and maintenance.
I have several candidate to consider:
C21C2, AC16, and Human Cardiac Myocytes (HCM) and iPSC-cardiomyocytes
Could you please let me know if you have any experiences working with these lines and if you have any suggestion or reference for me?
Thank you!
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Najmi La I hope this answer helps you in your studies.
It typically depends on the type of functional analysis you are planning for your studies.
If you are looking for cell lines that exhibit contractions, iPSC-derived cardiomyocytes are a good choice. AC16 cells don't exhibit contractions, but you can easily maintain them with good proliferation.
C2C12 is a skeletal muscle cell line. Although it exhibits contractions, this behavior is different from what we usually observe in cardiomyocytes because they will exhibit contractions only after forming into myotubes (after differentiation). I am not sure about human cardiomyocytes because it typically depends on age. You may observe some cell proliferation and attachment in the neonatal stage, but adult cells behave differently, and you need to maintain them in a specific medium and stimulate them to maintain functionality.
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The stainless steel bolt destroyed my front mass(AL7075) threads ☹️ any advice?
this is for an ultrasonic transducer
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Hello Ju Cheng ,
I am assuming that the destruction of the threads in the aluminum, AL7075, mass by the steel bolt occurs as a result of the ultrasonic tranducer being turned on. My guess is that the vibration from the excited transducer is causing the steel bolt to come loose, and scour the aluminum thread. Have you thought of using Locktite threadlocker on the bolt threads? Another possible solution is to install a steel insert, such as a Heli-Coi or other inserts, in the aluminum mass. In other words, pick a Heli-Coil whose internal thread size matches the thread size of your steel bolt, then drill and tap the aluminum mass to match the external thread size of the Heli-Coil. Install the Heli-Coil, break off the installation tang with a pin punch or cold chisel, then screw your steel bolt into the Heli-Coil.
By the way, is the threaded hole in the aluminum mass a through hole or a blind hole? The Heli-Coil would probably work best with a blind hole.
Here is the link for the various inserts:
Regards,
Thomas Cuff
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I am expressing two genes (CAR and a reporter) under EF-1a promoter in a lentiviral vector. I am using 2nd-generation lentiviral system (psPAX2 and VSV-G). My transfection efficiency in 293LTV cells was more than 99%. I collected the virus-containing supernatant, concentrated and then used it to transduce Jurkat cells. Unfortunately, my transduction efficiency has not exceeded 10% with MOI=10.
Here is my transduction in detail:
Polybrene (8 µg/ml)
Cells were washed once with PBS and then resuspended in base RPMI (FBS=0%)
Spinoculation (2400rmp, 2h, 32 °C)
The media was changed after spinoculation
The expression was checked 72h post-transduction.
I also tried higher MOI (20, 50, 100), but the expression decreased.
I appreciate any help.
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Likely related to your insert size. The larger your inserts, the lower your MOI. Every kb increase reduces your titer by roughly one half. You could scale up and concentrate your virus to achieve a higher MOI.
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Dear research community,
We have tried multiple times to transduce E0771 breast cancer cells with viruses to make these cells fluorescent or express luciferase. We have been very unsuccessful. If any of you has these cells transduced with RFP and/or luciferase vectors and is willing to share them with us, please reply back. We'll be more than happy to pay for shipping costs. Thanks to all!
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Hello, I would like to know if you were able to receive these cells. And if so, if you would be able to share them as well !
Best
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I have infected iPSCs with a lentivirus and sorted GFP positive cells. But after just a few rounds I am finding cells without GFP in culture. Had anyone seen this? Maybe cells are segregating inserts into only one of the cells after division? I think that this could be the problem, but never saw this before.
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What is the promoter? In our experience, If the plasmid has CMV or another viral promoter it tends to be silenced after a few passages.
Also, what passage are the cells? lentiviral cargo insertion is random in the genome, if the cells are early passage the epigenetics may also change.
Piggyback insertion and puromycin have been what worked the best for us.
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Let us consider that we are using an underwater acoustic transducer Model 630 in an underwater AUV application. Usually in the data sheets, the manufacturer provides the details of operating frequency, input poser, operational depth, typical receive response, and transmit response. Suppose I want to calculate the typical receive and transmit responses, then what formula is to be used?
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Such formula is relatively simple only in the case of a single vibrating piezoceramic plate with perfect circular shape and under the assumption that only thickness vibration is considered, and the results are measured on the symmetry axe. In typical transducers used for underwater applications it can be complicated and would require the knowledge about their construction and materials used. In complicated transducers (for example so called Langevin transducers) only quite sophisticated simulation can generate correct response, but not always very exact, since many elements cannot be perfectly defined (for example contact surfaces, glued elements).
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Hello everyone, please I am trying to transduce my suspension cells, I don't know if the transduction is successful so as to continue with the puromycin selection, please I will appreciate your kind response to this. Thanks
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If you have a GFP or other fluorescence marker on your vector you may monitor transduction efficiency by flow cytometry or fluorescence microscopy.
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Hi ,
I have been trying to transduce my iPSCs with sgRNA , at MOI 5 and 10 and I have had very less efficiency . (3-4%)
My Lentiviral supernatant is concentrated.
Could some one provide some insight on the transduction process?
Thank you
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Tom Masi : Thank you so much for the explanation . This has been really helpful .
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I am currently performing DNA extraction for a transduced cell with a virus but whenever I perform PCR, there is presence of viral DNA in it. So may I know if adding DNase into the cell before extraction and performing washes will eventually eliminate viral DNA and not the transduced cell DNA?
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First you have to protect your DNA of interest which you want to extract and then you can use DNAse to break down other DNA present in the sample. However, this is a bit tough to treat DNA like this.You can give a try if you find DNA purification ratio is ok then you can proceed on. Good luck.
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I would like to use puromycin selection in mouse primary cortical neurons to select for transduced neurons. Have anyone had any success doing it?
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Adam Figarski Planning of running a CRISPR screen in primary neurons and using puromycin selection for transduced neurons.
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I'm confused about 'homogeneity of cellular transduction' of AAV. Does this mean AAV can only transduce one certain type of cells after it enters into living organism?
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I believe that phrase is referring to consistency of the number viruses entering cells and the resulting intercellular expression levels.
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It seems to me that everyone just refers to J. W. Goodman's book "Speckle phenomena in optics: Theory and Applications". I agree this is a good book, however in my opinion there are some differences in ultrasound.
I would like to find answers to the questions:
- how realistic is it to assume that the ultrasound signal has a single spectral component (monochromatic light assumption in Goodman)? Is this assumption required?
- what is the effect of ultrasound transducer and the transformation from pressure to RF signal?
Thank you for your answers.
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Is it possible to transduce a human CAR into mouse T cells and have the mouse T cells function when the CAR binds it's antigen? I am thinking that if anything the singling domain of the CAR, as it is human, may be a problem for the mouse T cells.
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Thank you!
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I am designing an inverter circuit to produce 20khz, high current and high voltage power for an ultrasonic transducer.
I want to know what are the parameters in choosing a mosfet for my application?
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High voltage and high current are relative definitions.
How high exactly should they be?
Digikey shows 2 mosfets active in stock with 3300 Vds and 35A and 63A:
G2R120MT33J
G2R50MT33K
There is also a lot of choice in the 1700V ds.
The most important parameters:
-Rdson
-Gate charge/ Input capacitance
- Operating temperature
-Mounting type/ SMT or thru hole
- Vgs threshold
-and price and availability, of course
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can anyone help to me and explain in more details
I transduces mesenchymal stem cell with lentiviruse but it is not efficient...
/5 ml of concentrated lentivirus was added to each well of 6 well plat with approximately 100/000 mesenchymal stem cell and poroumycine selection was perfomed after 4 days, but was not efficient. it is important that concentrated lentivirus mixed to fbs free medium? I mix it to fbs+ medium and added to cells drop by drop
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Thank you very much for important points you mentioned , first I want to determine virus titration and second checked it with various ratio of viral concentrated
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How to calculate the amount of pressure generated by the ultrasonic transducer (single element or multi-phased transducer) after applying a certain voltage to it? I want to use this pressure field as the initial condition for longitudinal wave propagation simulation.
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Knowing voltage is not enough. You must also know the frequency (or the shape of excitating pulse) and the impedance of transducer material and the surrounding medium. Additionally you must know, that no plate is vibrating in a way as a piston, but in a more complicated way.
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I transduced my B cells with GFP containing plasmids using lentivirus and achieved 60% positivity. However, when I checked my cells under the confocal microscope, I can not see any GFP signals. (Maybe a little bit, but the signal it gave is similar to the signal given by non-transduced B cells). I was wondering does anyone know what could happened?
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Flow cytometer is much more sensitive than confocal microscope. I have several experiemce when a weak GFP-expressing cell is easily detectable by flow cytometer but direct imaging cannot see anything. Try increase your microscope laser intensity may help a little, but background will increase too.
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I am titrating lentivirus using Jurkat Cells. I transduced 250,000 cells with .05ul/.00005ml of undiluted virus in 1ml of RPMI media. We analyzed the fluorescence using Flow after 72 hrs (13.29%).
Which formula should I use?
Formula 1: (Number of cells transduced on day 1 x Percent fluorescent)/(Virus volume in mL)
Formula 2: ((Number of cells transduced on day 1) x (Percent fluorescent/100))/(Volume of undiluted virus Added (ml))
With formula 1, the titered amount will be: 664.5 X 10^8 TU/ml
With formula 2, the titered amount will be: 6.645 X 10^8 TU/ml
Formula 1 does give a huge titration number, but this is the formula I have found online everywhere.
Does this concentrated virus make sense?
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Colleaques calculations means titer (amount of virus) this can be calculated by by dPCR not immunoflorescient which posses ability to calculate proviral copies that have integrated in to target cells genome
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Dear researchers,
My research area is related to the development of metallic composites. I am switching to sensor development. Basically, I will work on the development of advanced materials for sensor applications. I need your guidance, suggestions, and ideas. I am also looking for collaboration in this area.
Looking for your valuable replies.
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We don't have any sensor development lab right now.
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Hi all, I wonder how it works in a Cre-dependent system when you use much less titer of Cre virus. Let's say we use titer 1 for virus A, and we use titer 1/10^3 for Cre. In ideal cases, we transduce X cells with A but only X/10^3 cells express due to Cre. But would it also be possible that there is less amount of Cre transducing each cell (resulting in the total transduced cell number > X/10^3) instead of the expected amount of Cre transducing fewer cells (resulting in the total transduced cell number =X/10^3)? If latter, would there be a difference between cell expression levels?
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Recombinant adeno_associated viruses(rAAVs) transcriptionally activated by Cre recombinase are powerful tools for determines anatomy and function of genetically defined neuronal types in transgenic Cre driver mice,while AAVs parvovirus from linear Single stranf(ss)DNA genomes packaged in icosahedral capsids so what happen depends on growth of AAVS!!!!!!.
More detailed in the attached ref.
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I'm trying to analyze bolted joints using PZT transducers. But "The electric potential will not be constrained for contact pair" warning is altering the results. How to overcome this warning?
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Thanks for your response. I'll look into it.
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I am trying to calculate the corresponding pressure from the received signal (measured in V) as a function of distance for an ultrasonic transducer.
I wanted to know is there any equation that correlates directly the corresponding pressure when I have the signal(V) measured by transducer. I am aware that the time echo method can be used to measure the delay in time and it is correlated with pressure. I however need to know the magnitude of the pressure.
Thanks in advance.
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Yes, Shadeeb, as Bernard rightly says, the manufacturer of the transducer should supply a calibration curve for the transducer relating the voltage measured into a specified impedance (e.g. 50 ohms) as a function of frequency, e.g. a curve showing for example 1.5 V/kPa at 1 MHz. Ask the manufacturer for this data, which should include accuracy specifications, e.g. +/- 15%.
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A horn antenna, usually used in connection with the UHF and microwave frequency range, is a transducer which operates on the same principle as the acoustical horn. Can we connect such antennae with the 377 Ohms that is identified with the impedance of free space?
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A horn antenna is usually well matched to 377 ohms at is mouth, where the impedance of the horn will be close to 377 ohms, and the input can be from waveguide or from 50 ohm coaxial cable. Inside the horn, the horn is designed to give a good transformation between the impedance of the input and the output over the designed frequency range, so that there is no significant reflection from the horn, typically less than 1% (-20 dB).
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Hi all, I am working with H4 cells that I have transduced with an engineered construct for a transmembrane receptor with a tag on it. I have detected the construct with immunocytochemistry and I would like to detect it via Western blot as well. I am using a different purified protein with the same tag as a positive control and have had no issues detecting it. However, I can't seem to find my target protein in my H4 cell lysate samples, and I am worried that the protein may be getting stuck with the debris pellets during my lysate preparation.
I am preparing my lysates with NP-40 lysis buffer, boiling my samples at 100C for 5 minutes before loading into the (4-20% gradient) gel, and have been transferring onto nitrocellulose overnight at 30V (at 4C).
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Yasuhiro Nishida The RIPA buffer worked, thank you for the help!
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Could you please say what would be the maximum output voltage obtained from the individual sources i.e., from piezoelectric transducer and electromagnetic converter
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My take is unless the output impedance of the two different systems are the same the current generated by one system will be hogged up by the other. Also the voltages generated by each system should be as much as possible (almost 100%) the same. However if the two voltage outputs are obtained in parallel then you see the voltage of only one of the systems (both have the same O/P voltage)but the currents adds up. This is what I would conclude with.
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The ultrasonic machine preferred is5Mhz capacity . The specimen used is Fiber metal laminate. Transducers using are Apprx.1 inch, so maintaining a focal length of 25.4mm between the specimen and the transducer. while performing through transmission pulse mode ,I have observed the echo transmission in the ominiscan are yellow in color, After placing the specimen the yellow color echo transmission are converted to green color. i have varied the Gain in the UT setting and threshold was set to 90%. even though green echo transmission are appeared on the screen . Also i am not able to capture the damage area of the specimen . The obtained image is attached .will any body help in resolving the problem i am facing to get the out put.
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The green echoes are just a display mode called envelope. This mode holds the signal on the screen. To turn it off i believe it is in Display > Overlay > A-scan > Envelope.
I am not 100% sure on your setup or what your damage mechanism is, but the through transmission method (2 probes, one on either side of the specimen) will only indicate discontinuities through a lack of response.
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Do we need a smoother thin film of Piezoelectric membrane material in the PMUT device performance? is there any dependence of resonant frequency on the roughness of piezoelectric membrane in PMUT?
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The resonant frequency depends on the geometry of the piezoelectric material. The thickness mode resonance frequency as such depends on the thickness of the specimen. If variation in the thickness of the film is not significant, you will get a clean resonance impedance response, otherwise the response will have kinks. The resonant frequency however will not change significantly even for the later case.
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I'm trying to make a stable HEK293T cell line that expresses GFP only in the presence of a specific unnatural amino acid (UAA). I packaged lentivirus particles containing my expression cassette, transduced cells, and selected for puromycin and blasticidin resistant cells (used two lentiviral constructs). Using 5 µg/mL puromycin and 6 µg/mL blasticidin, non-transduced cells were killed by 48 hours, while transduced cells remained viable. I expanded these cells and passaged 2x before assaying.
To validate my transduction, I set up an experiment in which I treat cells with or without a UAA and monitor GFP expression (which should only occur in the presence of UAA). When I perform this experiment, I observe that almost all cells are GFP-. Even more perplexing is that ~1% of cells are GFP+, but this phenotype is independent of the presence of a UAA (GFP+ cells without the addition of a UAA). I have attached microscopy images illustrating this issue.
My question is: how are my cells resistant to puromycin/blasticidin but fail to express my gene of interest?
P.S.
- My expression vector was constructed such that the UAA utilization genes are expressed on the same mRNA as the puromycin resistance gene, separated by an IRES.
- When I transfect my GFP/UAA utilization plasmids into HEK293T cells, they work as they should. This issue appears to be transduction-related.
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I think I have discovered the issue. I performed parallel transductions using lentiviruses packaged with mCherry to gauge transduction efficiency. With 293Ts, I identified a dose of lentivirus that yields >90% transduction. However, after selection, these cells remain unresponsive to UAAs and do not produce GFP. Thus, expression of the PylRS genes is likely not the issue here.
With these results in mind, I began to wonder if the 4 tandem repeats of tRNA cassettes would present issues during packaging or transduction. I hypothesized that, given the repeating sequences, this region of DNA may be subjected to excision via homologous recombination or some other process. To test this idea, I excised this region from my Pyl-tRNA-PylRS expression plasmid using restriction enzymes. Then, I transfected this DNA into the transduced 293T cell line that contains the PylRS genes but is unresponsive to UAAs. To my surprise, in the presence of UAAs and linear tRNA cassettes, these cells are able to produce GFP.
These results suggest that the repetitive tRNA expression module is somehow compromised during lentiviral packaging or transduction steps.
Thank you for your help Tom Masi, your feedback helped me arrive at this conclusion! I hope this is useful for anyone else experiencing this issue.
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I am looking for a technical guide for ultrasonic homogenizer design and manufacture.
I need a detailed explanation about how to design a transducer, booster and horn system for homogenizing solutions.
I would appreciate if you could please send me such a file or link.
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I want to compare impedance analyzer results with FEM ones.
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Monica La Mura Thanks a lot for your good response.
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I want to build a 100W 20khz ultrasonic transducer.
where or how can I find the dimensions?
my pizeo stack dimensions are: 50 17 6.5 mm
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Can anyone point towards a tutorial or video where I can perform co-localization analysis using ImageJ? I have a very simple experiment where I have stained HEPG2 cells with DAPI in order to count the number of cells and I have transduced a vector expressing GFP. I really just need to know the number of cells that are green in a given image. What would be the best way to do this?
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Hannah Bruce ,Chathura Abeywickrama ,
Johanna Marie Dela Cruz
, thank you for your replies. I will be sure to try your recommendations.
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We are trying to do some slice cultures and we need an established protocol for cultures. Also how transduce the slices with AAVs ?
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Hi,
We tried the method from this article and it helped!
May be could be useful for you , have a look
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I am using lentivirus system to transduce my transgene in T cells. but I observed transduction efficiency goes down over the day in T cells. While The gene is stably expressing in HEK293.
Can anyone help please??
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Andrew Lewis
...thank you for your reply. Since transgene is under constitutive promoter, hence at a snap time, the expression should be maintained.
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I need to design and simulate a power supply(generator) to drive 6 transducers. power of each transducer is 50 watts and they work at 40khz frequency.
can you please share a design of such generator with me?
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If you have never set up or repaired switching power supplies or converters, then I do not advise you to undertake the design and manufacture of a generator for an ultrasonic transducer. Try using ready-made DIY kits. Or, for the beginning, learn the schemotechnics of such ready-made kits.
Try to search "Ultrasonic DIY Generator"
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I am developing stably transduced HEK293T cells (lentivirus) with constitutively active RhoA gtpase. I have no experience in Microscopy and I want to characterize these cells visually and expand them prior to western analysis. I see this round morphology, but i am unsure if this is a bacterial/viral contamination or the cells are just sad. Are these just vesicles? These cells look different than HEK293T WT.
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Hi Diana, I re-plated the cells and they looked much happier. I even did a second round of puro selection, and the cells were robust. What size wells/plate do you use when you do the puro kill curve? I appreciate your assistance. Thanks.
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Hi every one
I would be very grateful for your expert help
Am planning for in vitro experiment in which an ultrasound waves ( frequency ranged between 1_50 MHz and pressure amplitude ranged between (0 1_5 MPa) will be applied to blood sample for a brief period (1 microsecond).
I imagine that I can do that by constrict a device as shown in the attached picture and which used in one study for ultrasonic plasmapheresis and which consists of a borosilicate glass capillary with a squared hollow to which a piezoelectric square ceramic, used as acoustic transducer, was attached underneath through a coupling hydrogel.
I don't Know if this is applicable? and if so, Is there a company that can installs it ?
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Can you cite the the article where you saw them performing ultrasonic plasmapheresis? Did this article mention what kind of equipment they used? How do you know that the pressure was 1-5 MPa, which, I think, is 10-50 Bars or Atmospheres or about 150-750 psi (pounds/sq-inch)? As Nikolay Pavlov mentioned, you probably cannot get those pressures using the output of a signal generator to drive your transducer.
Regards,
Thomas Cuff
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Usually SAFT is applied on shear waves using low frequency transducers. Shear waves provide very clean A-scan when compared with longitudinal waves. Due to large number of mode conversions of ultrasonic wave, multiple echoes can be observed in the A-scan acquired using narrow band longitudinal wave transducers. This makes it difficult for the implementation of SAFT technique.
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Yes but for implementing SAFT, TOF or arrival time should be known and in case of concrete, deriving TOF is a tedious task using longitudinal waves.
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There are two types of ultrasonic transducers: piezoelectric and magnetostrictive.
My question is:
Can the same power supply(generator) used for piezoelectric ultrasonic transducers be used for magnetostrictive ultrasonic transducers without loosing performance?
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Hello Ju Cheng ,
Perhaps the most direct way to drive the coil surrounding the magnetostrictive material would be to use a high power audio amplifier such as those used to driver moving coil speakers in a stadium. The input to the power amplifier could be a variable frequency sinewave generator.
I also found an interesting European patent that touches on your question, see attached document.
Regards,
Tom Cuff
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I want to make a magnetostrictive ultrasonic transducer for homogenizing applications.
I also want to make its power supply by myself.
I need some references (papers, patents, reports, books videos, etc.) that can help me in this way.
I would appreciate your help
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The ultrasonic piezoelectric transducer operates on the concept of the converse piezoelectric effect. When electricity is supplied to a piezoelectric material, it undergoes physical deformations that are proportional to the applied charge.
However, Magnetostrictive transducers are made up of a large number of nickel (or other magnetostrictive material) plates or laminations that are placed in parallel, with one edge of each laminate affixed to the bottom of a process tank or other vibrating surface. A wire coil is wrapped around the magnetostrictive material.
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I am currently working with recombinant lentiviral constructs to transduce/infect a mammalian cell line with also Polybrene at a final concentration of 8 micrograms per milliliter of transduction/infection media. After trypsinizing my mammalian cell line and centrifuging, I resuspended the cell pellet into media containing 1x pen/strep which I then aliquoted into T75 flasks containing premixed Polybrene and Lentiviral constructs. Will the presence of antibiotics affect the efficiency at which my lentiviral constructs infect my mammalian cells? Thank you very much for your time and help!
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For lentivirus production, yes. For transduction, i dont think so.
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I am using 2 ultrasonic assembly for cleaning purpose and I want to increase cavitation intensity.
(1) a ceramic transducer with a diameter of 30 mm and a horn with a end diameter of 8 mm.
(2) a ceramic transducer with a diameter of 40 mm and a horn with a end diameter of 8 mm.
Since the input power of (2) is higher than that of (1), I expected that the sound pressure of (2) is higher than that of (1), but it was not.
I think it is because the acoustic impedance of (2) is much lower than that of (1) (even though the power is high, sound pressure can be lower since Z=p/v is lower).
1. Am I misunderstanding something??
2. If not, how to increase the acoustic impedance of the ultrasonic assembly??
3. How can I estimate the acoustic impedance of the ultrasonic assembly??
4. What is the best?? the acoustic impedance of the ultrasonic assembly should be equal to the acoustic impedance of the media (water in my case) or as high as possible?
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However, If you look up matching layers in general, you will find that they are quarter wave (in the matching layer material) thick and of impedance (Zh*Zw)^.5, where Zh is the acoustic impedance of the horn material and Zw the acoustic impedance of the water (1.5 MRayls). Ideally, you should also have a matching layer from the transducer into the horn as well, same formula but using the impedances of the ceramic and horn material. The matching layer is designed for one frequency (where the matching layer is a quarter-wave thick) so will not help a lot for high bandwidth (very short) pulses.
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I think if the amplitude is large then the acoustic pressure also high.
I have tried to increase the acoustic pressure and use the ultrasonic booster which is known to increase amplitude.
But it did not work. The acoustic pressre measured by hydrophone was almost same as the acoustic pressure of the transducer without booster.
I am wondering
1. What is the relationship between the acoustic pressure and the amplitude.
2. What is the ultrasonic booster. Does it can increase the acoustic pressure??
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I think you may find what you need on the website of the transducer manufacturer, who may have application notes about the use of transducers, or the website of the horn manufacturer. They may all be optimised for water, or IPA or MEBK.
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Hi, does anybody know where to buy holders for air-coupled transducers with which the incident angle of the transducer can be adjusted? I searched online but didn't find anything even non-adjustable holders.
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To the best of my knowledge these arrangements are customized and are not readily available. And if available may not suit to your objectives. You contact any c-scan system providers. They may support in this regard.
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If I want to design an ultrasonic transducer or saw device, then what is the significance of the velocity which propagates through the medium in the form of energy.
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The design of ultrasonic transducer using piezoelectric material is related to wavelength and so ultrasonic propagation velocity.
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Hello Everyone,
I would like to mount ultrasonic transducers on a small cylindrical sample to measure P and S wave velocities (2" diameter and 4" length). Appreciate it If you can provide some references as a starting point on how to design such equipment, sizes, and specifications of ultrasonic transducers, etc.
Thank you
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Thank you very much, Dr. Ibrahim.
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I have an Bolted Langevin Transducer, resonating at its half wavelength frequency at around 52kHz, and the impedance measurement at resonance shows a 200ohm, and a -22° phase angle.
I check online, there are quite a number of different configurations of impedance matching circuit design proposals. I took the LC configuration, where the inductance L is place between the electrical source and the live wire of the piezoceramics, the capacitance is place between the output inductance and ground. So the circuit is simply a low-pass filter configuration.
The impedance measurement for the LC circuit + ultrasonic transducer demonstrates a 48ohm, -4° phase at around 51kHz.
However, when I powered up the device, with and without the LC matching circuit, using the same applied voltage input, the device without matching demonstrates an almost double vibration output compared to the device with matching.
Am I doing something incorrect? I thought bringing the impedance down to 50ohm will match to the electrical source output impedance, and an almost will ensure a maximal power transfer?
Looking forward to your feedback!
Thanks,
Xuan Li
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Hello Mr X. Li, I'm trying to design a matching network for a piezo transducer, it's easy to find a lot on informations for choosing a topology and calculating the value of the components, but my problem is: althought it's possible to build its own inductor, what kind of capacitor choosing for such a use? this information is never mentionned in papers, and I think that the frequency and also the power of the transducer should play a key role on the size of the device? thanks for your answer!
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Hello!
I have a question regarding my AAV experiments. I tried to transduce my HEK293T cells with AAV8. I already tested my vector with a gRNA in vitro and had a good knock-down rate, while also confirming the transfection with Lipofectamine 2000 via mCherry signal. Now I wanted to proceed with the production of my AAV8 vector. I checked for the intactness of the ITRs, produced them in HEK293T cells (triple plasmid transfection) and concentrated the AAV8 with PEG8000 and centrifugation steps before that, but did not finish the complete purification due to some technical hindrances (waiting for machines). I checked the viral titer with qPCR by using a standard curve of the same plasmid and a standard curve of a bought AAV8 virus with known titer. I transduced my HEK293T cells in order to check also their actual infecting titer and if they are working (I used a dilution series with a MOI ranging over 10 000). Even with a high MOI and a qPCR checking the viral titer via primers for ITR, I saw no fluorescence signal within the cells (72h incubation).
Suggestions on what could be the possible problem are highly appreciated.
Thank you in advance,
Nicole
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Thank you very much, I will try that!
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We are using a horn-type ultrasonic transducer for cavitation and cleaning purposes.
We experimentally found that the cavitation effect and cleaning effectiveness were decreased when input power was higher than a certain value.
The sound pressure measured by the hydrophone showed a periodic wave pattern when we applied proper input power, but it showed an irregular wave pattern when the input power is high (maximum value of sound pressure was high, but RMS value was low).
I have two questions
1. Does cavitation is promoted when the acoustic field shows a uniform and periodic pattern?? (even the maximum sound pressure is lower). Why??
2. Does the tip of the ultrasonic horn irregularly vibrate when high power was applied?? If not, why does the measured sound pressure showed an irregular pattern??
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Please read carefully the discussion https://www.researchgate.net/post/What_is_the_relationship_between_the_acoustic_pressure_and_the_amplitude_of_ultrasound as your question on the loss of cleaning efficiency when you exceed some intensity levels is largely similar...
At high level, the ultrasonic generator becomes largely non-linear meaning widening the frequency spectrum, including low frequency components able to induce unstable horn vibration, but also large fluctuations of pressure in the fluid and unstable cavitation bubbles size.
The only way to remain under control is to stay in the quasi-linear electric power/piezo-electric conversion/fluid coupling domain...
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I am using a Cole-Parmer 750-Watt Ultrasonic Homogenizer. Is it possible to control the frequency of the ultrasonic oscillation on this machine or do I need other pieces of equipment?
I am attempting to make translucent oil in water nano-emulsions following the protocol in this paper. My oil to water ratio will be significantly more however, it needs to be as high as possible while remaining translucent.
The energy setting of my homogenizer its in Joules.
So how do I convert from Hz to J ??
Here are the numbers form the paper but it doesn't make sense to be me because how can a higher frequency have a lower energy if E=hv?
20 kHz 44W cm^-2 = 440000 J
1.6 MHz 16W cm^-2 = 160000 J
2.4 MHz 7W cm^-2 = 70000 J
Here is their method
0.5 mmol of EDOT was added to 25 mL of aqueous solution containing 1.0 M LiClO4 in glass beaker cell. The 20 kHz ultrasonication to the water/oil mixture was conducted with an ultrasonic stepped horn (13 mm diameter, titanium alloy) connected with a 20 kHz oscillator (44 W cm−2, SONIFIER-250D, Branson Ultrasonics Co.) for 5 min. The sequential ultrasonication with 1.6 MHz treatment after 20 kHz was carried out using an ultrasonic transducer (16 W cm−2, Honda Electric Co.) connected with a Pyrex glass cylindrical tube (diameter, 24 mm; length, 75 mm) for 5 min. The further sequential ultrasonication with 2.4 MHz treatment after 20 kHz and 1.6 MHz was conducted by an ultrasonic transducer (7 W cm−2, Honda Electric Co.) connected with a Pyrex glass cylindrical tube (diameter, 24 mm; length, 75 mm) for 5 min.
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how can I calculate the power in watts from amplitude Bernard Garnier ier
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I want to find the resonance and anti-resonance frequencies of an ultrasonic transducer by analyzing its impedance.
so I need to buy a impedance analyzer or spectrum analyzer or something like that.
but my budget is limited.
do you recommend any device for my application and limited budget? :D
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If you want to measure impedance in a low cost way, get yourself
1) Suitable signal generator
2) An appropriately sized current sense transformer
3) a two-channel oscilloscope.
Measure the voltage and current as you vary frequency. Oscilloscope will give you the phase relationship between current and voltage across transducer. You can then calculate the real and imaginary components of impedance. I leave it as an exercise how you might calibrate this setup. Cheers!
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I want to apply a pulse-echo methodology (by using one single transducer as both transmitter and receiver). Do you know how to connect the transducer to the wave generator and oscilloscope to detect both transmitted pulse and echoes?
I have been trying to use the burst feature in manual mode by pressing the trigger button in the wave generator. However, when I do that, the oscilloscope is only capable of reading the wave generated instead of receiving as well the back-wall echo.
I am also using these types of transducers from Stemininc: Piezo Ceramic Plate 20x15x2.1mm 1 MHz, Piezo Ceramic Plate 7x7x0.2mm 250 KHz, and Piezo Ceramic Plate 20x15x3mm 710 KHz. Thus, I'm not sure if they are indicated to use this pulse-echo methodology.
I have two alligator cables connected to the oscillator and to the transducer: 1 to work as the transmitter and one to work as the receiver; and they are both connected in the same wires of the transducer. However, so far, it seems that these alligator cables connected to the same transducer are giving me the same wave. 
Do you know if this equipment as it is is capable of doing these readings:
  • reading of the transmitted and received waves (of amplitude vs. time), separately, by using this setup as is (when using two different cables), or
  • reading of the transmitted and received waves combined in the same curve: maximum peak sent and resultant received echoes (back-end wall or cracks, for example)?
I am using a manually triggered pulse sine wave of amplitudes of either 10Vpp or 24Vpp, but the outcome is always identical. Should I be using a higher amplitude to make sure I receive the echoes?
Any help would be much appreciated.
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You basically need a T/R (Transmit/Receive) switch between your signal generator and your transducer, and your oscilloscope and your transducer. When your signal generator is producing the outgoing periodic pulses used to drive your transducer, the T/R switch disconnects the oscilloscope from the transducer. While your transducer is receiving the reflected signal between the periodically generated pulses from the signal generator, the T/R switch connects the oscilloscope to the transducer, but disconnects the signal generator from the transducer. Note, be aware of the concept of range ambiguity, i.e., if the reflecting surface is further away than the round trip time between two of the signal generator's pulses, then the incoming reflected signal may fall between the next two pulses of the signal generator making the reflecting surface's distance appear shorter than it actually is.
How you actually realize your T/R switch will depend on your drive signal amplitude, and the input impedance of your oscilloscope.
Regards,
Tom Cuff
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I was maintaining modifed (transduced) A549 cell line. They are looking strange Either they are stressed or got some sort of contamination. There are lots of big irregular sized ball like structure, but they are attached. I have tried increasing serum percentage and every possible things that I know but it didn't help me. I have attached a picture, which you may find useful. Thank you.
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S. Béatrice Marianne Ewalds-Kvist can you tell what are those big yellow almost round things?
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Good day All,
OEM recommendations for the avoidance of gas/air bubbles are to install the echo sounder and speed log transducers between 1 and 5 degrees in the transverse direction. Has any tank analysis been performed to verify this recommendation.
I have reviewed Dr. Klaus von Bröckel's article "Echo Sounders versus Air Bubbles in Research vessels' on the Hydro International website that that article does not include the figures that are referenced in the article.
If the vessel includes a bulbous bow will installing the transducers between 1 and 5 degrees athwartships closest to the bow help mitigate the effects of bubble sweep down. Installing a keel box is not acceptable for speed KPI considerations.
Thank you very much and kind regards,
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@ Bernerd Garnier. Thank you very much for your insight and for suggesting a reasonable approach to optimally locate SPDL and NES transducers. Your comment to ensure a 'very smoth and accurate profile continuity' is genuinely appreciated.
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I have two IDTs (interdigitated transducers) orthogonal to each other o a piezoelectric substrate. I want to know what happens when an RF signal is applied to both the IDTs at the same time. how to find out the orthogonal interference of two acoustic waves?
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The article says that high intensity waves are non-linear. If waves are non-linear they interact with each other. If they are linear, then that do not interact with each other. This means that they each behave as if the other is not there.
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I have transduced MCF7 cells with a lentiviral vector containing a tetracycline inducible expression system, but my protein is not expressed in MCF7 even though I selected with puromycin.
I have successfully used the same lentivirus in other cell types, but every time I use it in MCF7 cells it fails.
The puromycin resistance is placed very closely to the protein I wish to express so I don't think the protein insert is lost in the vector?
Are there any suggestions or explanations for this?
Thank you for taking the time to read my question!
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I have encountered the same problem recently with MCF-7 cells. They should transduce easily. Did you figure out what was the problem?
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I have used both viral supernatant as well as viral particles concentrated by ultracentrifugation to infect EA.hy926 cells. But I have been unable to transduce them. The same viral particles have been able to efficiently transduce other cell lines. I have tried both 8ug/ml and 10ug/ml polybrene, but have not been able to transduce the cells. Can someone please help me out?
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What is your measure of transduction success? Transgene expression, surviving rate of stable cells during antibiotic selection? May be it is not the transduction itself, but something else.
Have you tried different MOIs and repeated transductions? Some cells need higher virus concentration.
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is it possible to make a DIY impedance analyzer for checking the resonance frequency of high power ultrasonic transducers? for example a face mask welding ultrasonic transducer
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Yes, it is possible.
The most straightforward way is through Ohm’s Law. By using a voltage sensor to measure the test signal's voltage across the transducer and using a current sensor to measure the test signal's current flowing through the transducer, together with various signal processing algorithms, the impedance over the frequency range of interest can be obtained. The test signal can be produced by a signal generator. As an alternative, you also can use the Inductive Coupling Method for the measurement of the impedance of the transducer. More information about these methods have been detailed in my following papers/monograph:
[1] Z. Zhao, "Measurement setup consideration and implementation for inductively coupled online impedance extraction," Ph.D. thesis, Nanyang Technological University, Mar. 2021, doi: 10.32657/10356/146738.
[2] Z. Zhao, A. Weerasinghe, Q. Sun, F. Fan, K. Y. See, "Improved calibration technique for two-probe setup to enhance its in-circuit impedance measurement accuracy," Measurement, 2021, vol. 185, Art no. 110007.
[3] A. Weerasinghe, Z. Zhao, N. Narampanawe, Z. Yang, T. Svimonishvili, K. Y. See, "Single-probe inductively coupled in-circuit impedance measurement," IEEE Trans. Electromagn. Compat., 2021, doi: 10.1109/TEMC.2021.3091761.
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Hi all, I transduced my K562 cell lines with letivirus and use puromycin as a selection marker. When I did the cell viability curve, WT K562 only die at a concentration as high as 90ug/ml, which I don't think is a normal concentration. Unfortunately, when I select my transduced K562 cells using 90ug/ml, all the cells died. When I decrease the concentration to 30ug/ml, the selection didn't work well. Anybody knows what happend to my cell line?
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Hello,
The optimal concentration of puromycin suitable for selection of resistant mammalian cell clones depends on many factors like cell line type, culture medium, growth conditions and the quality of puromycin used. So before selection, the optimal puromycin concentration needs to be determined by performing a Kill Curve titration.
So for the Kill Curve for puromycin you can use the concentration range of 0 - 20ug/ml puromycin on normal K562 cells. If the WT 562 dies at a concentration as high as 90ug/ml it means that puromycin is less potent. So, try using another fresh vial of puromycin. This may help.
Best Wishes.
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I am doing primary fibroblast cell culture and transduced my cells with a lentivirus with 10^7 TU/mL. I added 7.5 uL of virus to about 250,000 cells to have an MOI of about .3. Of note polybrene was also added at 8uL/mL. When the antibiotic kill curve was done all of the control cells dies but only 1 percent of my transduced cells died. I understand that adding polybrene will increase my transduction efficiency, and given that the 10^7 TU/mL number was generated without polybrene, my actual TU/mL might be higher when used with polybrene. Regardless, I don't think polybrene would change my transduction efficiency from about 25-30% (MOI .3) to 99%. Additionally, my cultures had odd morphologies and a reduction in proliferation that are not expected to arise from my vector (just a promoter-antibiotic resistance-T2A-dCas9).
Does anyone know what the issue might be?
Thanks!
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Hi, when you have lots of resistant clones they degrade the antibiotics so the concentration is lowered and non transduced (non resistant) cells can survive (this effectively happen easily with G418). It is also possible that during selection the resistant cells proliferate and fill the plate: how do you calculate your 1% ?
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What will happen if the frequency response of the displacement transducer is too small in the explosion tests of civil structures? Does it lead to relative time distortion of the displacement history curve? Or the structural displacement responses would be incorrect?
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I think this depends on what you want to achieve to measure (temporary of permanent effects). Besides the horizontal, vertical displacement I would recommend to include as well the Tilting effects of the structural members.
Prior to the actual measurement I would recommend as well to model the impact and asses the prelimanary results. The measurement would then be a calibration exercise.
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Hi all, I am using PZT to excite the beam structure and measure the impedance using dynamic signal analyzer. And I also build the finite element model in ANSYS to do harmonic analysis and get the impedance data. My current work is to update the simulation model based on experimental measurements. I read some papers, but all use trial-and-error method. I am wondering if there is an efficient method to conduct model updating.
If make the whole FE model in an optimization loop, the harmonic analysis takes really long time for each iteration. On the other hand, there are too many parameters, and most of them have influence on impedance curve. So the parameters to be updates are also undetermined.
Any suggestions or references are appreciated, thanks.
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Dear Yang Zhang ,
I think you can reduce the parameters of the system if you used a physically based model for the response to the acoustic waves. There is an electric circuit equivalent model that mimics the acoustic system.
In this model the mass, the elasticity constant, and t he dimensions of the objects are the parameters of the model.
This is just a proposal of an electronic engineer to solve such problem.
Best wishes
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Hello,
I recently came upon this phrase in the Addgene webpage describing lentiviral transduction.
"Typically cells transduced with lower dilutions of the virus will have higher levels of expression. Consider expanding populations transduced with a variety of dilutions and pick the population that has the most desirable level of expression."
I couldn't understand this, as to my thoughts, higher dilutions of the virus should result in bigger MOIs (more lentiviral particles and more lentiviral genome copies integrated per each cell) and thus should result in higher expression. I am currently troubleshooting lentiviral transduction to BJ fibroblasts, trying out different viral titers.
Could anyone give me an explanation on this?
Thank you.
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High quantities of virus could lead to cytotoxic side effect,therefore in some cases it may be better choose a artifacts due to non^ healthy cells.
MOI for Lenti virus range from 1 to 30.MOI describe number of virus particles needrd to infect one cell according to special equation for more detailes see attached ref.
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We are looking for a reliable provider or vendor, that could provide us a technical and financial offer for piezoelectric transducers.
These transducers will be utiliezed for energy harvesting applications and experimentations.
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I want to simulate a sono-reactor.
Can anyone share a reliable simulation file for simulation of a power ultrasonic transducer and horn in ANSYS or COMSOL with me please?
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dear Hakan Kandemir they doesn't include some analysis like modal and harmonic analysis