Transducers - Science topic

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?

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?

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

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

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:

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!

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?

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.

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?

do you know if it’s possible to generate and detect ultrasound with electromagnetic transducers on lead (Pb)?

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!

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.

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?

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?

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).

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

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.

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

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.

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?

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.

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?

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).

And the (1) EGFRvIII-mCherry U251 cells were also tested negative using 2-step staining (https://www.emdmillipore.com/US/en/product/Anti-EGFRvIII-Antibody-clone-DH8.3,MM_NF-MABS1915-25UG + https://www.thermofisher.cn/cn/zh/antibody/product/Donkey-anti-Mouse-IgG-H-L-Highly-Cross-Adsorbed-Secondary-Antibody-Polyclonal/A32766),

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?

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.

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?

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!

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

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.

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.

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

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?

No longer available

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.

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!

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.

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!

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!

The stainless steel bolt destroyed my front mass(AL7075) threads ☹️ any advice?

this is for an ultrasonic transducer

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.

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!

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.

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?

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

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

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?

I would like to use puromycin selection in mouse primary cortical neurons to select for transduced neurons. Have anyone had any success doing it?

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?

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.

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.

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?

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

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.

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?

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?

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.

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?

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?

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.

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?

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).

Could you please say what would be the maximum output voltage obtained from the individual sources i.e., from piezoelectric transducer and electromagnetic converter

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.

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?

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 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.

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.

I want to compare impedance analyzer results with FEM ones.

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

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?

We are trying to do some slice cultures and we need an established protocol for cultures. Also how transduce the slices with AAVs ?

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??

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?

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.

Hi every one

I would be very grateful for your expert help

I don't Know if this is applicable? and if so, Is there a company that can installs it ?

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.

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?

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

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!

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?

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