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I did a water-soluble extract from a fruit and I'm going to use this extract to treat cells in vitro.
In order to test the highest dosage in vitro (2 mg/mL), I will need to use a large volume of extract dissolved in the medium for the treatment. What is the maximum volume I can use in vitro (dissolved in culture medium) which will not disturb the cells and cause their death?
Initially, I put 30 ul of extract in a total volume of 150 ul (20% of extract in medium). Is that too much and eventually can cause cell death? Does anyone have that experience?
Thank you.
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If at lower concentrations it had no effect, I always add a solvent control at par, in this case you could use sterile water if your extract is aqueous and do the viability test and the result would show that the extract is the one with the activity.
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Hi, I have problem to sterilize freshwater plants (Anubias, Bucephalandra). I used chlorine salts and ethanol. I got contaminations. I think AgNO3 could works. Do you have any other protocols for sterilization?
Thanks for all responses.
Bohuš
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Here's a basic protocol I've used in the past for plant samples
5-20% bleach (need to optimize for your tissue) with 0.5% detergent (triton or tween or even dish soap), incubate with gentle agitation 2-10 minutes (you'll need to optimize this step too). Then rise 5 times with sterile water.
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Hi guys, Is it possible to cultivate interesting mycorrhizal fungi in vitro with in vitro cultivated plants or roots? If somebody has better skills, give advice please. What plants should I use ? Thanks for all responses and advices. Bohus
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As per the INVAM website and literature chicory root will be the best whoever the technology has been tested using roots of many crops like carrot, tomato....
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I'm currently working on anther culture of tomato and most of embryos I've got usually ended up looking glass-like structure, that makes they are so hard to continue their development into normal structures.
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Thanks for the answer Max Winkeljohn , Alberto Tosca I'll try your recommendation.
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I am currently taking care of Plasmodium falciparum in vitro culture and in order for me to keep them alive, I was wondering how and when should I feed them if you have any protocol to advice me. I have access to complete media and RPMI.
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You can use the method suggested by Silvia Di Santi. You should change the media every 24hr. If the parasitemia is high, split some and keep it below 5%, we usually keep it around 2-3% unless we want to perform any antimalarial assay. Most importantly, you should check the parasitemia level using microscopy every 2-3 days intervals at least and provide fresh RBCs while splitting. For higher parasitemia, you can use inactivated serum or plasma from AB+ donors.
Below is our article for a brief explanation and synchronization method as well:
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Dear Good People
Is it possible to study the effect of a compound on the activity of a certain signaling pathway but you only have antibodies for the total protein/nonphosphorylated form involved in that pathway! or it would be a waste of time and money?
"P.S."... No abs for the phosphorylated forms are available📷
Thanks in advance!
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Dear Doaa Yamani,
you could try to do immunoprecipitation experiments with, e.g., anti-phospho-Y or anti-phospho-S antibodies, to pull down all phosphorylated proteins in your samples. Then do western blotting and use your antibodies for the nonphosphorylated proteins to detect any enrichment after compound treatment of your cells.
There are several commercial kits available, but in principle it comes down to incubate your cell lysate with the first antibody that will bind to all phopsphorylated proteins in your sample (PY-20 detects phosphorylated tyrosine). Then you capture that complex with, e.g., Immunoglobulin binding Protein A or G coupled to either beads or agarose. After washing the pellet, resuspend your sample in sample buffer for western blotting, the bound proteins are released and loaded on the SDS gel. Detection is done with your antibodies for the nonphosphorylated proteins you mentioned in your question.
You have to be careful though during the detection step, since the Immunoglobulins (the heavy and light chains) used for the pull-down experiment can cause problems during the second detection step (check the species your antibodies are derived from to minimize cross-reactivity!).
Good luck with your experiments,
Christian
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I was wondering whether NAD+ (not nicotinamide riboside or nicotinamide mononucleotide) can enter cells when supplementing the medium of a neuronculture with NAD+
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Hello Fa Ro
You may please refer to the research article attached below. It will be helpful.
Best.
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Good day,
i have a general question about tissue culture.
I have found the following recipe for Epipremnum Aureum "Marble Queen":
Leaf Explant: MS Medium + 4.54 µM TDZ + 1.07 µM NAA (Thidiazuron in Micropropagation of Aroid Plants by Chen and Wei (2018), p. 105, DOI: 10.1007/978-981-10-8004-3_4)
Specifically, I have the following questions.
1) Do i only need to autoclave the agar with distilled water (I use a pressure cooker for this) and when the agar has cooled down a bit just add the MS, TDZ and NAA and mix it or do i need to autoclave the MS as well?
2) Will the TDZ dissolve in the agar water at all and how hot can the agar water be to add the MS, TDZ and NAA?
3) Is it even necessary to autoclave the water incl. agar (in the pressure cooker) if I clean all the jars with NaClO (sodium hypochlorite)?
Thank you in advance!
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In general, all things associated with tissue culture need to be properly sterilized. For me, I autoclave the complete media (MS, hormones(I use 2.4-D, NAA, BAP, Kinetin), and agar) along with the culture vessel (petri dish or test tube). But it is better to filter sterilize (.2 micron) the hormones and vitamins (of the media) and add them to MS media (agar mixed) when the temperature drops to about 50 degrees celcius.
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Hi anyone can help me to find methods to increase the multiplication of garlic plants in vitro culture. I am using Ms media, NAA and BA solution atm?
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Dear Maduka Wehella have you read the paper from Bhojwani (1980)? Inside you can find many key tips. Consider genotypic factor: it's more important than nutritive formulation; hormones and doses, at least in my experience. Besides other reports, also check E. R. Joachim Keller and Angelika Senula Micropropagation and Cryopreservation of Garlic (Allium sativum L.). Be patience, garlic seems easy but it's not. Good luck!!!!!
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Various plant growth regulators were found to have a positive effect on organogenesis and somatic embryogenesis, but which one is more important?
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Dear @Reza Ghahremani
Different plant species, such as C. canephora, A. thaliana, and Musa spp. responded successfully to the Somatic Embryogenesis induction using different explants, conditions, and concentrations of PGR. The details can be accessed at:
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What you see in the image is dry extruded faba bean which hasn't been exposed to any treatment (heat or chemical) after the extrusion. The DMEM has been added 12% FBS, 2.5% TMP/SMX, 1% Pen-Strep og 1% Fungizone. This is the result after incubation for 24hr at 37°C.
I know that the DMEM changes color after pH-changes, but I don't understand why it would change into two separate phases and what it is in the extruded faba bean that causes it. I spread it out on TSA-plates and it didn't show any growth of the bacteria that was previously there (B. cepacia and R. insidiosa) or any other bacterium, so the sterilization was deemed effective.
I appreciate any answers and theories.
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DMEM typically contains a pH indicator (phenol red)- the media change its color depending on pH - yellow below pH 6.8 to bright pink above pH 8.2. You could check ph in your media. Above are some discussions. Good luck.
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SLOW GROWTH STORAGE for the mid-term conservation of a large number of species, including tropical and temperates. In this case, reducing the in vitro growth through the application low temperatures and less hours of light.
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The StarPac bags are sealed and held for 2 weeks in a growth room at 25 °C with a 16 h photoperiod provided by fluorescent lights (40 μM m-2·s-1). Then the StarPac bags are transferred at 4°C in low light. You can work with 12 to 16 h of photoperiod.
Best Regards,
Jean
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Anyone can help me with this trouble shooting of plasmodium falciparum in vitro culture? I've been tried to culture plasmodium for approximately 2 months but they didn't grow well. Everytime I check thin blood smears of my culture, they appear like this (see the picture I attached) . It seems they couldn't invade or infect the red blood cells and stay outside. Please give me some advice about what I have to do so they can grow well and the ruptured schizont can invade RBCs.
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Please what did you later do to get the cells to grow? Please how do I also get P.falciparum culture? I want to test the effect of my research drug on the growth of plasmodium cells in vitro
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We know that somatic embryo can be used as an artificial seeds, but encapsulation include many kind of material. I ask to researcher to give their experience for us.
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Take a look at this video link
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Hi,
In order to measure the LDH level in the experimental groups in Pc12 cells, I tried the lactate dehydrogenase activity assay kit (MAK066-sigma). I used one on the brand's page for the protocol, but the results were meaningless. There are some studies that used the same kit but different protocol. I don't know what to do. Is there a protocol that uses this kit or that you can recommend?
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I suggest using your LDH Positive Control (or another known high LDH concentration) and serially diluting it to give you 4-6 concentrations (check the kit sensitivity as no point going below it more than once). Test all these in duplicate.
The results from that should tell you where the problems are. e.g does the signal decreases in a linear line? Is there is a large variance between duplicates? That will reveal much information about any method and tell you if it's worth continuing with. If it's not linear around the concentration your expecting, or the variance between duplicates in larger than would give you meaningful results to compare what you want, you need a different method or you need to perform it better. Some of these things do get better with practice as well! Good luck!
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Hello to everyone! I am trying to multiplicate a specific variety of Kiwi with tissue culture. I managed to multiplicate some. Some of them came from callus and some were produced directly from the starting material. What I would like to ask is, do the plants that come from callus differ genetically from the mother plant?
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The cytogenetic and genetic stability of the regenerated plants is one of the key prerequisites for efficient clonal propagation. Plants regenerated from relatively undifferentiated callus cultures possess a vast array of genetic changes. Such variations can result in useful agricultural and horticultural products. But sometimes, the variations in traits other than those of interest may be undesirable. Anyhow after regenerating plantlets, you can go for genetic fidelity assessment to understand the level of genetic variation.
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My experiment involves treating recipient cells (at 70% confluency, 24 hours after seeding) with EVs (adding EV suspension dropwise to the culture media) before assessing a specific miRNA expression in the recipient cells after 24 hours (by qPCR). I see no change in miRNA expression in EV treated vs untreated cells.
From a separate set of experiments we know
i) EVs are taken up by these cells (using dye based method)
ii) EVs are enriched with this specific miRNA
Any suggestions would be greatly appreciated!
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miRNA of interest is not expressed in the recipient cells and I saw a robust overexpression with miRNA mimic transfection (qPCR) in these cells. ISH sounds like a pertinent method to try. Thanks again!
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Many pieces of researches have shown that responding explant percentage, embryo number, plant regeneration and also response rapidity increase in TCL explants compared to larger size explant. Now, I ask all researchers to share their experiences and reasons.
Thanks
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Minimal plant tissue offers many advantages over big explants. In case of TCL, cells are directly in contact with the nutrients, and have maximum chances of organogenesis and somatic embryogenesis on optimized growth regulators.
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We know that light has a key role on different plant mechanisms, but its effect on the embryogenesis is question. Thanks for expressing the opinions of researchers.
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Yes, the nature of the color and its intensity can affect somatic embryogenesis experiments and may result in many differences among the obtained explants.
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Due to the fact that silver ion is a blocker of ethylene receptors, some studies have reported that the accumulation of ethylene has decreased during in vitro culture due to their application.
How can this be justified?
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Actually, silver ion is a blocker of ethylene receptors, competing for the same subtract, so I guess that in these in vitro studies the accumulation inhibitory effect is indirect, given the autocatalytic nature of ethylene.
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Fungal and Bac contaminations are common in vitro culture. However, Ppm works alright with some of the contaminated plants . But bleach and ethanol doesn't work at all. Any recommended solutions or media types?
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Meristem cells in shoot apex are devoid of pathogens like bacteria, fungi or viruses.
Suggest meristem tip culture to obtain pathogen free plants you are working with.
For details of this procedure read plant tissue culture book.
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We are planning for wet-lab experiments in India, so we need a few chemicals reagents. The cost of products is comparatively less in ChemFaces company based in Wuhan, China. Can this company be trusted? will it provide high-quality and authentic chemicals. Your suggestion will be of great help!
Website link of ChemFaces
I am waiting for your valuable reply...
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Sir,
To the best knowledge of me, this company has a wide range of chemicals, some of them can be seen in perfect articles in some well-known journals, such as Cell.The products from ChemFaces, China is OK, but you have to make sure that you are contacting a qualified and honest dealer or the correct contact information of the manufacturer.
Good luck!
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Dear colleagues, fellow scientists and students,
I am working on establishing of protocol for BioID in plants. I am aware of already existing protocols such as cell culture, the transient transformation of tobaccos and root culture. For our purposes, we will need to work with seedlings and I was wondering if someone ever tried to perhaps transfer the seedling onto solid MS media enhanced with biotin. Could that work? What would be a good concentration to start with? I'd be grateful for any insights or links to relevant literature backing up the yes-no argument.
Thank you and all the best in your research!
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Tomás María Tessi Thank you for your input!
That is exactly what we wanted to do initially however we need to do a treatment to plants that should be 24-48h and I do not think the plants will survive it. Or they would be so stressed that the whole experiment will be biased.
So I am thinking about alternative methods. But perhaps I can do the treatment and transfer them to the liquid MS+biotin as biotinylation itself is 2-6h.
Thank you and good luck in your research too, maybe I just needed to look at it from different angle :)
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The use of antibiotics, preservatives and similar chemicals is aimed to be avoided in cultures. Since isolations without the use of antibiotics and preventive chemicals is my goal, latent bacterial and sometimes fungal contamination is encountered. Does anybody has experience treating source plants with any preventive chemicals or antibiotics few days or weeks prior to isolation to reduce or eliminate the internal contaminants?
Any recommendations/suggestions will be highly appreciated.
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Just be sure to use sterile water for any rinses, no point in doing all the hard work to try and get surface sterile plants just to add back contaminants from non-sterile water.
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Hi, guys, I am a postgraduate with lots of experience in culture contamination. However, when I was about to be sure I will never be bothered by contamination, this (as is shown in the pic) occurs! and spoil my almost one-month effort........I dont know what this is and where they may come from, can anyone give me some help?Please!
Thank you guys for your answers! After several days of observation, yellow objects grew more in number, and there are more and more dead cells in the culture. Some objects are floating while others in inserted between cells. From my point of view, they do not seem like cell debris or any other "healthy" things..........
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Here is another possibility. It might not be contamination with an organism or virus. When you disperse your cells with trypsin to subculture there may be some small clumps of cells passed to the new flask. Incomplete dispersal. The cells in the clumps die but do not affect the growth of the other cells. I would have to see the actual cultures to be sure, but make sure that your trypsin/EDTA (or whatever you use) is new.
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Although it's not a very effective sterilizer alone for plants but still being part of treatments in all researches. i wonder over its widely essential use and what factors decide the time of ethanol treatment?
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Dear @Amina Ilyas You may find a detailed discussion related to your query at the link given below as to why 70% ethyl alcohol is used for surface sterilization:
I would like to add that I am fully convinced with the explanation by @Yuan-Yeu Yau on that thread.
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Since tween 20 is no doubt an excellent surfactant to remove surface tension and provide better exposure of plant surface for sterilization, by removing the bubbles which are more problematic in case of plants having plenty of hairs and trichomes. I wonder if in case of unavailability of tween 20, can a common detergent or dish soap provides the same benefit or there is any other cheaper compound as a replacement?
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Tween 80. Maybe Triton-X-100. Especially Tween 80
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Hello! I want to isolate RBEC from a rat, I used the protocol - https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4208856/. However, I used 5-day old rats instead of adult rats. Subsequently, the addition of puromycin (4 μg / ml) killed 100% of the cells. Could this be due to differences in the age of the rats, or is it worth looking for other errors?
Attached is a photo of a mixed culture, no puromycin.
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Dear Georg!
Please You look at the article and protocol:
Culture of rat brain capillary endothelial cells This method has been adapted from previously described techniques (Roux et al. 1989; Szabo´ et al. 1997; Deli et al. 2000). Briefly, 2-week-old Wistar rat cortices were dissected free of meninges and minced. All animals were treated according to protocols evaluated and approved by the ethical committee of INSERM, France. The dissection was performed on ice and in phosphate-buffered saline (NaCl, 137 mM; KCl, 2.7 mM; Na2HPO4,8mM; KH2PO4, 1.5 mM). The cortices were cut into very small pieces (1 mm3 ), homogenized with a 5-mL pipette and digested in a mixture of collagenase/dispase (270 U collagenase/mL, 0.1% dispase) and DNAse (10 U/mL) in DMEM for 1.5 h at 37C. The cell pellet was separated by centrifugation in 20% bovine serum albumin/DMEM (1000 g, 15 min) and incubated in the collagenase/dispase mixture for 1 h at 37C. The capillary fragments were retained on a 10-lm nylon filter, removed from the filter with endothelial cell basal medium supplemented with 20% bovine plasma-derived serum and antibiotics (penicillin, 100 U/mL; streptomycin, 100 lg/mL) and seeded on 60-mm Petri dishes coated with collagen type IV (5 lg/cm2 ). Either 4 lg/mL puromycin was added for 2 days or 3 lg/mL puromycin for 3 days. Puromycin was then removed from the culture medium and replaced by fibroblast growth factor (2 ng/mL) and hydrocortisone (500 ng/mL).
Of course, the more cells are obtained from a younger animal, the more sensitive they are to antibiotics.
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After in Vitro culture of my sweet potato callus, how do I sample or prepare the callus to be subjected to UV-A radiation and also calculate the dosage used.
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You subculture in a liquid medium and keep on a rocking surface so that the clamps will separate to give you individual cells. You need to find the LD 50 and the GR 50 in order to establish the optimal dose for your callus by exposing to varying doses or different exposure times.
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In media preparation, the plant tissue culture media needs to be adjusted to ± pH 5.8.
Why it is so and what would happen if the pH is lower than 5 or more than 6.5?
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As mentioned already by the RG members following this thread, pH influences the availability and uptake of nutrients and plant roots' growth.
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I need to examine the in vitro antiviral activity of a drug in the presence of a series of dilutions of
human serum up to 40 percent (e.g., 5 percent, 10 percent, 20 percent, 40 percent).
An EC50 value for 100 percent human serum can be extrapolated from these data and the serum-adjusted EC50 values reported. In addition, I need to determine EC50 values in the presence of physiological concentrations of α-acidic glycoprotein and human serum albumin.
What will be the difference between the data from the first paragraph vs the second?
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Hi,
Differences will be related to different affinity and binding of different drugs for albumins and α-acidic glycoprotein. If your drug has a higher affinity for α-acidic glycoprotein than albumin then differences in concentrations of α-acidic glycoprotein may be important for a free fraction of the drug in vivo. It should be always recognized for what fraction of plasma proteins your drug has high affinity. It's especially important in the case of lipophilic drugs and for drug-drug interactions related to concurrence for the same proteins.
Best regards,
Tomasz
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Dear all,
I am performing Caco2 permeability experiments using transwell inserts and I am using phenol red permeability assay to verify the monolayer integrity.
What is the maximum phenol red permeation allowed in order to state that there is no damage to the cell monolayer?
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I honestly can't recall what we used. The manufacturer should give this info. One wants a pressure-equalized scenario to avoid solvent drag in either direction, but if the monolayer is relatively tight, then the volumes are less important and 1.0 mL on the BL side should be fine.
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Hello, colleagues.
I had this problem in my in vitro cultures of Cnicus benedictus, which I derived from immature embryos. After several months of vegetatively multiplying the plants to get a lot of clones from each genotype, which I further want to use for polyploidization with oryzalin, I have experienced endogenous bacterial contamination. The senior doctor, who is a specialist in in vitro culture in our department, says that some embryo cells may already have had endogenous bacterial contamination, and now that I've cut the plant tissue while propagating, we've come across the cells, and the contamination has gone to the media. Does that seem likely?
I use 100 microliters per 100 milliliters of PPM (Plant Preservative Mixture) and, I'm recommended to increase this concentration three times. If that doesn't help, I'd have to use the media using antibiotics, but I'd like to avoid that because it would take me at least a week of passage and another week of passage back to classic MS after a week.
What would you recommend that I don't have a similar problem in the future? Alternatively, what are your pointers for handling the current sticky situation?
Many thanks in advance for the suggestions and good day and good health.
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You may need to do regular subculturing. Or you may use non infected plant parts of your in vitro grown plants to start fresh culture. Using antibiotics may affect the culture. You may also think of starting the culture through callus induction. You may later check the genetic homogeneity of the plants using molecular markers.
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3D Cell Culture is newly optimized and promising discovery in cell organization research and other researches that needs evindences in cell culture (in vitro) level. Are 3D Cell Cultures mimic epithelial cells because they are lining up the specific surfaces ? Is it possible to create an cell culture environment as we want in 3D cell cultures ? (e.g. microenvironment that show inflammatory responses as a result of cancer-related inflammation). Is real-time screening of cell response (e.g. after drug exposure) available in 3D cell cultures ?
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Thank you for your contributions to my question. It has developed my point of view. (like always) I wish good look to you too, it was nice to be your colleague :)
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I'm studying the immune profiles of persistent/ chronic infections in human PBMCs. I'm interested in knowing if there is any published ex vivo or in vitro model that allows me to do repeated viral stimulation to mirror the potential persistent infection in human?
I know there are humanized mouse and organoids, but they don't seem optimal. Thanks a lot.
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One of the main issues in better acclimatization of in-vitro woody plants is "stem thickness". How can we get the ticker stems during multiplication stage?
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Not only the light intensity but also the the quality/source of light influence the growing shoot..for example:BLUE light strongly inhibited the elongation. Incandescent light promoted both growth of stem diameter and elongation of leaf petioles (From:Reference+my own experience)
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I need a protocol to detach osteoclasts from in vitro culture
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Hello Sonia, very good question. But I don't have idea in this field.
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What is the best way to solubilise palmitic and oleic acid respectively ?
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I do not know what for you want to use it, but if for cytotoxicity, this paper seems like something what can help you :)
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Seed coating agents and their results
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We cover the seeds of various crops with metal nanoparticles (trace elements) for better germination, development and increase yield. https://www.researchgate.net/publication/281065009_Stimulation_of_Seed_Viability_by_Means_of_Dispersed_Solutions_of_Copper_and_Silver_Nanoparticles
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Doing an in vitro study with nanostructured lipid carriers with vitamin D and with just vitamin D alone, and just trying to figure out the optimal incubation time so I don't stop it too late having saturated the cells and wind up seeing no difference.
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You may have a look at the literature below for nanostructured lipid carriers with vitamin D
For vitamin D alone incubated in Caco-2 cells, I recommend 10 nM 1, 25D3 for 24 hours as a physiological condition, or you can try 100 nM 1, 25D3 for 24 hours to check the maximal changes.
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Hello everyone,
I would like to observe the effects of a recombinant protein on in vitro cultured cells. For this reason, I need to know a concentration at which recombinant proteins can be used in in vitro culture without killing the cells. I've been looking for information but I couldn't find a proper answer to my question.
Did anyone ever work with recombinant proteins incubated with cells in vitro?
If so, does anyone have any tips about working concentrations, at least a suggested starting work concentration, from which I can start?
Thank you in advance to you all!
Have a nice day
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I worked quite a lot with rh and rm proteins in vitro. The answers depend on the source of the protein if you are purchasing the recombinant protein make sure that it is carrier-free or at least that the carrier not harmful to the cells. Many cheap rh proteins sold as control proteins (i.e. ELISA, WB) are usually not useful for cell culture application due to preservatives such as sodium azide. Then as suggested by Simon Caulton you need to determine an effective and non-toxic concentration of your protein to the target cells. Another important question is how the cells are supposed to interact with the target protein. In the case of the cell membrane proteins with the known receptors is fairly easy to get meaningful effects by simply adding the protein to the cell culture media. For many proteins that are located within the cytoplasm or nuclei, however, as well as proteins that are subject of activation or posttranslational modifications this approach is usually not effective and overexpression by plasmids is preferable. The direct delivery of the protein to the target cells by lipofectamine is possible, but usually not effective due to the harmful effects of the transfection process to the cells that usually mask the effects of the target protein.
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We are testing the anti-inflammatory activity of these compounds on human PBMCS in vitro. Is there any recommended concentrations that are being used? I would be grateful for some references in that respect. Thank you in advance
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Interesting question, carvacrol is the subject of my doctoral thesis
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1.Technologie des Légumes (Y. Tirilly - C. Bourgeois) Tec Doc 1999 - ISSN : 0243-5624 : Génétique et création de nouvelles variétés de légumes (M. Branchard - M. Pitrat) 27-44.
2. Variability in plants regenerated from tissue culture (E. Earle - Y. Demarly) Praeger1982- ISBN : 0-03-059364-6 :In vitro culture of barley: a method to study Rhynchosporium scald disease and select plants resistant to the toxin, rhynchosporoside (M. Branchard) 343-350.
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Michel Branchard you can scan the two papers into electronic files first. Then you upload the files into ResearchGate.
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For certain reasons, frozen tissues are only for in situ staining. Is it possible to use frozen tissues for other purposes? Such as isolating the tumor cells from the other cell types present for in vitro culture. I value any feedback and discussion on this.
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Hello, Many methods exist. Cancer cells have unique mechanical properties that can be tested all-mechanical or optomechanical. Also, cancer cells can also be identified visually by shape or by optical scattering.
If you have the possibility to measure cell hydration is also a way for differentiation. The cancer cell always has higher hydration rate than normal cells
Goog luck!
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if we put nanoparticles inside cell culture media, is it possible to measure when the particles uptake the cells? can DLS measure the particle inside the cells?
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No, you should use transmission electron microscopy, assuming the particles are electron dense enough to be seen.
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I am a student working on bamboo tissue culture (in vitro).
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Add kanamycin (10 μg/ml) to medium for 10 days and transfer them to fresh medium without antibiotics.
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When determining subculturing time, what markers such as whether the explant shoots are green, callus size, etc.?
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We are working to propagate some citrus rootstock in vitro. We noticed that subculturing every 4 weeks is better in growing plants because the medium of cultivation during this period is depleted. However, the period of subculture depeded on the plant species and culture conditions.
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Is there a current/reliable way to isolate mouse islets (keeping their morphology and viability for culturing in vitro) without perfusing the pancreas through the bile duct?
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Mouse islets are commonly used in diabetes-related studies. Adequate amounts of good quality islets are prerequisites for a reliable investigation. Three major manipulations are employed in the islet isolation procedure: in situ pancreas perfusion with collagenase, pancreas digestion and islet purification. The whole procedure takes 30-45 min for each individual mouse. By using this protocol, a reasonable number of islets can be obtained in a relatively short period of time. This protocol has been proven to be practicable and reproducible. It can be easily followed by individuals who do not have previous experience in the related research field.
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seed pre-disinfection and tetrazolium test for select viable seeds to seedling in in vitro culture.
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thank you Alyona M. Alexandrova I will try that.
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  • I want the method please
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Kindly go through this useful PDF attachment.
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I am working on alectoris chukars' semen in in vitro condition. I want to know dilution rate of its semen for evaluation of semen traits such as motility, viability and etc. Note that I use beltsville poultry semen extender (sexton extender) as semen diluent.
thanks in advance
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thanks for replying dear Wassan m.hussen
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I want to investigate the effect of some nanomaterials on the plants using in vitro culture. So it's important to make solution of those nanomaterials to use in MS media with different concentration. The nanomaterials I want to use are MgFe3O4 and MoO3.
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Required amount of Nanoparticles you can simply add to your culture media, or you can make stock solutions of Nanoparticles in sterile water and dilute according to your need to add in your culture media. In each case ensure that your nanoparticles are throughly dispersed. Ultrasinication is good option for mixing properly. In some cases Nanoparticles unique characters may get degraded due to heat, so preferably donot autoclave the nanoparticle solution. Add it after autoclaving the MS media. Nanoparticle solutions can be filter sterilized separately.
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Hi,
Normally, the success of seed germination through in vitro culture is influenced by several factors such as medium composition, seed origin (plant species), seed maturity...
The success to ensure normal germination and development from in vitro cultured seeds of perennial lignified plants is very limited. Why??
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Well dear Mohamad Issaoui , of course the need is to clone any of these plant species. However, in the starting question, there is no information as to WHY germinate seeds under in vitro conditions. To do what? They don’t germinate at all under in vivo conditions? Species rescue?
However, if ever it is to clone them, the worst thing that one can do is to germinate them. And that’s where the difference between Angiosperms and Gymnosperms come in the discussion. Any Angiosperm sp can do something after germination, what is not the case for Gymnosperms. And ontogeny has nothing to do in the story. It is a question of physiological age. Yes, physiology is the expression of genes through biochemistry, cytology, morphology, etc. Nothing new! Except for geneticists!
What is the point?
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I am working on Papaya micropropagation where I am getting plenty of shoots but they are not increasing in height. I have tried diffuse light, GA3 but did not see improvement. Please suggest a better treatment.
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Dear Parth Desai .....
you can decrease the Light intensity for a week in first sub culture, or also use low concentration of GA3, that will effective in elongation of your new shoots
Best regards
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I am working on production of stevioside from Stevia rebaudiana under in vitro culture conditions. We want to check the cytotoxicity of the plant extracts on cell lines, preferably pancreatic cells. Please advise and is there anyone who can provide us with the cell lines in Delhi
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I am looking for a drug that can stimulate TGF-beta expression of epithelial cells in vitro culture. I appreciate your suggestions.
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Hi, guys
I`m quite fresh in the field of culturing CD8 T-cells in vitro and I need an advice. I want to test if a population of CD8 T-cells may give protection against LCMV-Armstrong virus infection in mice. So, we want to isolate the cells from LCMV-immune mice, expand, then transfer into naive hosts and challenge the mice with LCMV. As a measure of protection we want to use viral titer. We rather doubt if the cells are protective.
The point is that antigen specificity of the overall cell population is not known. The cells of interest contain some antiviral effectors, but relatively few and it`s not known whether these effectors play any role in the response.
TCR-transgenic mice are unavailable as far as induction of the marker I`m studying is not possible to achieve.
So, I`d avoid antigen-mediated CD8 T-cell expansion as far as it can promote some cell while depleting the rest from the population of interest. Initially I planned to use IL15-mediated expansion. Preliminary tests with that cells from naive mice were promising for 50 and 100 ng/ml of IL15. Cells grew very fast and within 1 week I was able to get 5-6 millions cells starting from 0.1 million of sorted cells.
I am making a preliminary trial with total memory CD8 T-cells from LCMV-immune mice. The cells grew quite fast on IL15 and expanded more than 20 times within 1 week. They were viable, proliferated in response to LCMV-peptides and secreted IFNg.
I wanted to expand them more to make some additional tests, but after 1 week of culture in vitro the cells stoped growing. Right now 1 week passed, but they hasn`t expanded even a little. They are just alive, neither die, nor proliferate.
Unfortunately, after stimulation with plate-bound aCD3e (1 mcg/ml overnight) that IL15-expanded CD8 T-cells from LCMV-immune mice died very fast. So I`m not sure if I can use this method.
Any ideas how to expand them? Should I try weak aCD3 stimulation with plate-bound antibodies and cytokines? Or I`d rather try some stimulator cells with soluble aCD3? Or some cytokines? IL15/IL15Ra?
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Correct...
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Hello all! I am currently facing a big trouble with fungi contamination in in vitro culture of native forest seeds, because they come from field collection with a lot of endogenous contamination (fungi). The disinfestation with alcohol (1min) + 2% hypochlorite (15 min) does not work, they insist to proliferate ... Would pre-treatment with fungicide or some other solution solve this problem? Any suggestion?
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Hi,
As an undergrad I used to make calli from tree buds coming from outside and I know contamination can be a huge problem. We used the following method: 1min. 96% ethanol, 5min. HgCl (not sure if it is allowed everywhere although it is not volatile or anything and as long as you don't ingest it , it is fine), 3x5min sterile water. I think for seeds ethanol can be left even longer. As you already know that you have a lot of fungal contamination you could also treat with a fungicide prior to the actual sterilisation.
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Hi,
I am a second year graduate student and have been doing flow cytometry on embryonic neurons as a part of my experimental procedures. The neurons were cultured in vitro and allowed to form synapses for 10 days after which I used them for flow cytometry. In order to understand the percentage of live and dead cells, I used 7AAD as a marker. Ideally, I would have expected two distant peaks indicating 7AAD positive (dead cells) and 7AAD negative (live cells). However, my graph has three peaks.It looks like a negative peak and two positive peaks. I am however a little confused about this and wanted help to interpret this data.I am attaching the histogram plot in order to get an idea of what it looks like. If anyone has any inputs please do comment. I would really appreciate your help.
Thank you.
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If in your conditions neurons strongly attach to the substrate, the method that you use for harvesting neurons will cause cell damage. I think that it is likely that you have early and late necrotic cells, because late necrotic cells show a more damaged cytoplasmic membrane and incorporate higher amounts of 7-AAD compared to early necrotic cells. Also, it could be cellular aggregates. To be sure, first discriminate aggregates (FSC-A vs FSC-H) and then you can make a FSC -A vs 7-AAD dot-plot . Late necrotic cells will be smaller and with higher fluorescence for 7-AAD, respect to viable 7-AAD negative cells. By contrast, if what you are obtaining is a cell cycle profile of the dead cells, as Waqas suggests, you will find that cells with a higher fluorescence for 7-AAD tend to be bigger. In any case, FSC -A vs 7-AAD dot-plot will allow a better discrimination of live and necrotic cells because you will analyze two parameters instead of one.
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I came across the two contradicting views that ROS levels must be low under low oxygen (microaerophillic) or in absence of oxygen (hypoxia).
Other researcher shows that ROS levels are high due to inhibition of ETC. Generation of superoxide anions by NADH dehydrogenase shown to play role.
I wish to knew what happens under physiological conditions in tuberculosis inside granuloma or macrophage where conditions are hypoxic or similar ?
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Dear Samsher,
Unfortunately (or luckily), ROS are complicated and a collective name of many different reactive molecules. I don't work on mammalian systems but plants. Similarly, ROS bursts can occur upon entering hypoxia, but may decline (depending on the type of ROS) upon reaching anoxia depending on the type of ROS and organelle/tissue studied.
I would always suggest looking at their methods and check how and when they did their measurements and draw careful conclusions from there.
Best of luck
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for last two months I try to culture WB-F344 cell which was purchased from JCRB cell bank. As mentioned in the documents I cultured them with MEM including 10% FBS, but after 4-6 passage the cells were died.
any suggestion ?
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thank you.
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I am student at FUM University in Iran is working on establishment of in vitro culture of pomegranate plant (Punica granatum) and has problem with Plant Growth Power. The plants produced in the stage of Proliferation are very weak, with thin stems and narrow leaves. How can we enhance Plant Growth Power (pomegranate plant) in vitro culture?
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What do you mean "in vitro culture" in pot or cal in Petri dishes ?
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I need information about biostimulators for seed germination, plantlet growth and plant development of following medicinal plants:
1. Nigella sativa,
2. Trigonella foenum,
3. Coriandrum sativum,
4. Rhus coriaria.
Also, I would like to have some papers about development of stress tolerance of these plants both in vitro and in vivo conditions.
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Abstracts:
1- Effect of diluted sea water on growth, yield, essential oil productivity and chemical composition of coriander (Coriandrum sativum L.) plants.
2- Efficiency of chemical, biological fertilizers and gibberellin on Coriandrum sativum L. under the condition of salinity and calcareous soil.
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I'm trying to optimize the NRU assay on 3T3 cells as low OD540 reading (about 0.1-0.2) were obtained from previously done experiments. I prepared 50ug/ml stock and incubated it at 37C for 1.5 hr, added to cells and incubate for 3 hr.
- I took the neutral red solution that's incubated at 37C from waterbath into the hood prior to washing of cells with PBS. Therefore, I'm thinking if the solution might get a little bit cool down (less than 37C) while I was washing the cells with PBS and that caused precipitations.
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Yes, they look like neutral red crystals, try heating stock to 40-42°C for some time instead of 37°C, followed by 0.45uM filtration. It may help.
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Hello,
I work in cancer immunotherapy particularly in CAR T-cells. I have leukaemia cell lines such as KO-52, ksaumi and THP-1. They express my target antigen at different levels. My CARs are also active, they kill ovarian tumour cell line expressing the same antigen but not leukemia cells. I am wondering what are the possible reasons for tumour cells resisting killing in vitro.
Thanks
Abed
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There could be many reasons for lack of in vitro CTL tumor killing:
HLA or H-2 mismatch
Leukemia cells may lack TAP
Down-regulation of MHC on tumor cells (check with labelled Ab)
CTL lack perforin or Granzyme B expression (check by Flow Cytometry)
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Hi
The inhibition of spore germination and growth of Botrytis cinerea upon treatment with toxic molecule occured on solid culture medium (petri dishes) as predicted before. Strikingly, the spores germinated normally in liquid medium that contain also the toxic molecule at the same concentration (flasks).
How can we explain such observation??
(See attached pic.)
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I know there are metabolic pathway shifts in fungi when they are grown under those two conditions. The fungi may be inhibiting uptake of the inhibitor, inactivating the inhibitor or may just be insensitive to it under growing conditions as a submerged culture.
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Hi everyone,
I am a first year PhD student and I am working on a protein called Thymosin Beta 4 (TB4) and using it exogenously to treat injured cells in the kidney called podocytes. I am mainly working in cell culture right now and I am treating the podocytes with TB4 by adding it to the media that the cells are cultured in.
One recurring question I keep getting asked is how do I know that the TB4 is entering the cells? I have seen results when TB4 has been added, so I know that it is definitely having some sort of an effect. Does anyone have any idea of how I could prove that the TB4 is entering the podocytes? Any help would be greatly appreciated.
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Hi again, if the exogenously added TB4 is somewhat different from the endogenous (tagged, labeled with a fluorescent probe) you might still consider localization experiment.
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I want to do the in vitro culture of the fish pituitary under different treatment condition. So, can anyone kindly help me the protocol for the pituitary culture.
Thanks,
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Dear Rakesh,
I want to collect the pituitary gland of catfish in normal laboratory condition after fully acclimatized for the in vitro experiment.
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we have done red cadamba seed germination in vitro by treating with fungicide and double sterilization with 50 % Clorox and denatured alcohol and inoculate it in MS media with hormone but it shows low germination rate even after a month .
please advice me on how to increase the germination rate and reduce the contamination rate
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Hello
Are your seeds alive ? Which hormone and which fungicide are you using ? Why using MS medium rather gelled water or moisted filter paper ? Is there any seed dormancy for this tropical tree ?
Regards
MJ
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Rooting problem.
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Half strength MS plus different combinations of NAA and IBA
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I'm interested in how the secreted protein MMP-2 might be affecting a certain type of cell. I thought about adding the recombinant MMP-2 protein to in vitro cultures of the relevant cell type at different concentrations, but I can't seem to find any literature describing assays that do this so am not sure how best to approach it. Does anyone have experience of using metalloproteinases in this way?
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Not sure I understand your question. Are you asking for possible outcome measures of an in vitro exposure to MMP2? if so, based on the type of cells you're culturing, you can look at tight junctions proteins (e.g. Zo-1 and claudin). These tight junction proteins are degraded by MMP9 and 2 (e.g. see ).
Also, the link below is a publication where the effects of MMP9 was evaluated in an in vitro model. It may give you some ideas on how to proceed. https://www.nature.com/articles/s41598-017-16250-3.pdf?origin=ppub
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Dear researchers,
What are the black structures that appear on Botrytis cinerea mycellium after two weeks of in vitro culture??
I tried to observe them under binocular microscope and I think that they are fruiting bodies but I am not sure.
Can anyone cofirm this ??
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Hi, These are sclerotia , The fungus produces highly resistant sclerotia as survival structures in older cultures.
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Thank you for your assistance.
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Please go to:
You will find all protocols for malaria research.
Kind regards
Christian
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There are various techniques for in vitro culture of various tissues and also cornea.The success for corneal culture across world is still in primitive stage as far as I knew. Then what is your method and at what stage you are.
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Dear Dr Rao
At present we are industrialising a bioreactor dedicated to long term storage of human corneas. By restoring intra ocular pressure (among other things) it allows increasing the viability of endothelial cells (+23% after 4 weeks of storage).
Results will be soon published.
Sincerely yours
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I am working on wheat mature embryo callus regeneration..Facing problem with regeneration..Plz suggest me suitable regeneration media(India wheat variety)...Please help
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Dear Nisha
Aydin et al. (2011) reported that the highest plant regeneration from wheat mature embryos was observed in MS medium supplemented with 12 mg/l dicamba + 0.5 mg/l IAA.
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