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In Vitro Cell Culture - Science method

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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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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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I have planed for animal experiments in India. We need a chemical reagent for our experiment, the cost of it is less in ChemFaces, Wuhan, China (https://m.chemfaces.com/) and the cost is slightly high in ChemScene (https://www.chemscene.com/). Which company among these two is very authentic, provides high-quality chemical compounds? Which chemical vendor can I place an order from?
Your suggestion will be of great help. Looking forward to hearing from you all
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Hi,
I applied on PC12 cells 100 ng/ml NGF and incubated for 72 hours to differentiation. After 72 h, approximately most cells differentiate and then I applied an anticancer drug but all of the cells including the control group died. The medium I used while applying the drug is the same as the medium I used for differentiation. I couldn't find my mistake. I'm waiting your idea or advice.
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By control group, do you mean cells grown the same condition but without the cancer drug?
You need a no drug control under the same conditions as the drug but without drug. And yes the control cell must have high survival.
It is possible that extended serum starvation induces apoptosis in these cells. It is critical that you know the survival tendencies including plating density and resistance to serum starvation induced apoptosis.
Understand that serum "starvation" often refers to the time since fresh serum (media change).
Cells are supposed to die without serum. The more "normal" a cell line is, the more sensitive it is to serum starvation.
72hours with no fresh serum in many cell lines increases apoptosis without any other treatment.
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I have been isolating RNA from in vitro cell cultures with some success following a basic chloroform extraction followed by isopropanol spin and ethanol washes. Due to the amount of samples collected at a time, however, I am only able to run a couple and need to freeze the rest. I have been freezing them at -20 C for a short period of time, but it seems that when I try to collect RNA from these samples they give me a low 260/230. Are there any special procedures or tips for isolating RNA from frozen samples?
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I have tissue biopsy punches stored in trizol at -80. Can anyone share the protocol to isolate RNA from such samples. Is there a possibility to isolate DNA, RNA and protein in same sample with trizol?
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Suppose  i seeded 5000 cells in RPMI medium, so how i decided that how much volume of RPMI medium for 5000 cells treated.
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I am currently doing a clonogenic assay on MCF-7 cell lines. Please how do I determine the volume of extract to add to my 6-well plate already containing 3 ml of my media+ cell suspension?
I need urgent answers cuz I need to add extract after 24 hrs of seeding the cells.
Thanks in anticipation of reasonable responses.
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I seeded RKO in a 96 well plate at a density of 5,000 cells/well, 200 uL of complete media in each well. 24 hrs later, I removed all the complete media and add my treatment. I incubated the cells in the treatment for 24 hrs. Cells in the well were still alive. I added 10uL of MTT Reagent (25mg/5ml of Assay Buffer) to each well and mixed on an orbital shaker
I added 10uL of MTT Reagent (25mg/5ml PBS) to each well and mixed on an orbital shaker.
The cells were checked at 4 hours. There were formazan crystals formed. There was no color formation when I first added 100 uL of DMSO. From my friend's advice, maybe the DMSO was too little so I added another 100 uL. This time, there was color but instead of purple, it was dark blue!
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I understand this has already been stated; however, I wanted to recapitulate the advice of using freshly prepared reagent.
I've been performing the MTT assay on a variety of cells over many years, and my recent anecdotal experience, this discussion board, and some pub-med and google surfing led me to believe that the use of reagent which had been frozen could have been the source of the issue (hear me out!).
I prepared the reagent as instructed by the manufacturer (Thermo-Fisher) ~1-2 weeks ago, filtered, and made 10 mL aliquots to store -20*C wrapped in in aluminum foil. I thawed the reagent to use in my recent treatment time course (incubating MTT for 3 hours), but noticed there were some bright blue production among the purple formazan crystals.
I also noticed some precipitate which had crystalized onto the 15 mL conical tube, and stayed there regardless of light vortexing and even pipette scraping.
I've seen the blue production before, but as a PhD student, I did not think twice about it (my previous lab, MTT was always stored in -20*C).
All that said, I think that the preparation, storage, and care taken during use could contribute to the differences.
Although TF and other companys' protocols state that it may be stored for ~6 months in -20*C, I believe the freeze-thaw conditions could cause these differences. The colors produced by the formazan dyes will vary depending on the tetrazolium salt used as a substrate for the reaction; therefore, if the salt precipitates or alters during storage, it could consequently lead to altered outcomes.
I'm not saying that this is 100% your case, or even mine for that matter, I just wanted to provide a little insight from my perspective!
Thanks!
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I'm in need of a reference or a method to come up with a protocol that allows one to get the protein-adjusted EC50 for an antiviral drug in presence of human serum albumin and acid glycoprotein.
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Hi Min, you can check a recent review like this
and the references therein. Best: Karoly
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Hello, I am new to biofilm research and have a question about growing and measuring biofilms.
We want to research the effect of a type of enzymes in breaking down biofilms in showers, baths, hot tubs, and hot springs.
Therefore we must devise a way to grow and measure biofilm that is close to natural biofilms in showers etc.
Are there any existing protocols or any other researches that may be useful?
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Dear Nanami, the following papers maybe helpfull to find answer to your question.
You can isolate bacteria from surfaces and than you can characterize their biofilm formation capabilities on different materials surfaces like stainless steel or polymer surfaces. There are both quantitative and qualitative methods to evaluate biofilm formation.
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How do i measure the enzyme activity of human GBA in cos 7 cells that have been transfected with mutant plamids. After 24 hours of plating cells in a 12 well plate, I transfect cos 7 cells with pcDNA containg the GBA cDNA with the specific mutation. Transfection is done using Lipofectamine 2000 following the standard protocol with 2ug of plasmid DNA. After 48 -72 hours of transfection the cells are harvested and assayed for enzyme activity using the SIGMA GBA enzyme activity assay kit(MAK 129). How can i extract the proteins from the cells without denaturing it? I used 50mM phosphate buffer to extract the cells and then centrifuged. The supernatant was used for enzyme assay, but the activity was very low even in the wild type one..
please suggest how i can improve this assay.
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Try harvesting the cells 24-48 hours after transfection. Hope washed the cells three times 1X PBS or the buffer used before harvesting, check the pH. Also, use ice-cold buffer, and perform centrifugation at 4 degrees. Check the protein content in the supernatant.
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Do you know any normal mouse lung cell lines other than MLE 12 (which transformes Tumorogenic)?
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Hi Robert,
I found that Wi38 is a human lung fibroblast line, not from mouse. It is mentioned in the article you attached and also in the Sigma Aldrich website from where I wanted to get it.
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What is the recommended seeding density for H9C2 cells (specifically in a plastic tc t25 flask)? We have been seeding at the recommended 10x10^4/cm^2 (250,000 in t25) and they are reaching ~70% at 48 h instead of 72 h. The majority of our cells seem to be attaching. Can we seed at lower amounts without changing the cells? Additionally, how many cells would be in a confluent t25? We are using the media and trypsin specified by ATCC.  
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I observe ~48hrs doubling time and seed cell @ 10K per cm2 usually need to split every two to three days.
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I'd like to learn more about the "biological action" of MRSA in bovine mammary gland and because of that I looking for in vitro (cell culture) studies related to that.
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The last couple batches of RPMI 1640 media I have prepared have not been the correct color after the addition of FBS. When aliquoting small volumes before the addition of FBS, I have the expected pink color however once the FBS is added (10%, 50 mL into 500 total mL) the color is a faint red. This color change most likely signifies some kind of pH change, but I'm wondering if the FBS is causing it and why now but not previously? 
The media has no contamination, I've left it at room temperature and in the incubator overnight and found no growth. 
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I have also exactly the same problem (different color previously and now).
can you help me by telling me if there was any problem in the cell growth in culture done using this medium (faint red color medium)?
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My project is to develop an in vitro cell culture model by dissociating a fresh salivary gland biopsy, culture the cells and perform viability assays using flow cytometry every 24 hours.
My question is whether it is possible to store the fresh intact biopsy for about 12 hours before I start the dissociation procedure. Do you think that will have a negative effect on the cell viability afterwards?
Thank you!
Emma
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Sure, if you perform this method in any case via the same procedure (12 h at 4°C) the results you get should be comparable but may differ from other data published in different papers /(using different protocolls, procedures).
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Capsicum annuum seeds were placed in MS media 5.8 pH on February 6th, no significant changes can be seen so far. The seeds were planted via in vitro and the containers placed under white germination lights at room temperature (around 10 to 30°C).
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@ Luis Fuente Cevallos,
1. Make sure that when you sterilize the seeds with EtOH or/ bleach, you don't sterilize them too harsh, which can kill the seeds. Follow a good protocol from an good journal.
2. You can grow some seeds in soil to see whether they germinate or not.
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Hi,
I work with an inflammatory model of Caco-2 cells. I seed the cells on transwell filter.
I establish the inflamation by adding the 10ng/ml of IL-6 in basolateral medium. I dont know this dose is enough any sugession?
After 6 hours exposure to cytokine I treat the apical part with my samples for 4 hours. I want to decided which way is best to check the anti inflamatory responses to my sample.
Which one of the following sugesstion is the best based on protein expression and inflamatory stimulation?
1. 10 hours IL-6 stimulation and in parallel 4 hour treatment with samples and continuing IL-6 stimulation for 14 hours more (total 24 hour) and ending the expriment and collect the sample?
2. 10 hours IL-6 stimulation and in parallel 4 hour treatment with samples and end the expriment (total 10 hours) and collect the sample?
Any better sugesstion and why?
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It was based on a response curve for a active phase protein.
A digested samle.
looking for inflamatory profile.
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Hi
The most researchers used 50µM of ex-527 for treatment of cells. How can they get to this concentration, whereas IC50 of this drug is 89nM ?
I want to use this drug for Jurkat cells and molt 4. . How can I find the best concentration of ex-527 that inhibit SIRT1?!
MTS assay is good?!
No article exist setup this drug for Jurkat and molt4.
Thanks in advance
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IC50 for a compound/inhibitor for a cell line depends on several factors, including the cell line in question, solvent for the inhibitor, culture conditions, and proposed endpoint for the treatment. Therefore, it needs to be determined empirically under the desired conditions for the proposed endpoint (when known). If a study has used much higher concentration of the inhibitor for a similar endpoint, it could be due to due to other variables (cell line and culture conditions etc). It is always better to find out an optimum IC50 before performing an experiment.
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can 293T cells form laminin clusters in vitro in cell culture? or can such cluster formations and ECM formation only occur in vivo?
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Hi An, by definition, ECM proteins that are not soluble are integrated into the extracellular matrix.
It does not matter if this occurs in vivo or in vitro.
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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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I need a protocol to select heat tolerant cell lines or callus that i can use to breed heat tolerant ornamental plants.
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Enhancement of Reproductive Heat Tolerance in Plants
April 2015PLoS ONE 10(4):e0122933
DOI: 10.1371/journal.pone.0122933
LicenseCC BY 4.0
John J BurkeJohn J BurkeJunping ChenJunping Chen
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Recently, I started differentiating mouse neurospheres towards noradrenergic neurons (the procedure takes 5 days with the addition of GDNF and BDNF) and towards the GABA neurons. In both options cells are cultured in 5% O2 at 37*C.
In the case of NA neurons in vitro cell culture looks fine when it comes to the number of cells.
However, while differentiating towards GABA neurons with more compounds like purmorphamine, IGF-1, forskolin or VPA, after a few days of cultivation, many cells are detached (plates are covered with ornithine/laminin) or undergo apoptosis which results that after 2 weeks of differentiation it remains very few cells in culture. Is this a normal process in this direction of differentiation?
I use protocols based on these articles: 10.5966/sctm.2014-0083 (two-step protocol) and https://doi.org/10.1016/j.celrep.2015.04.056
Could you give any advice? Thanks in advance.
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It seems to me that GABA and compounds which mimic it are toxic to neurons. You can try decreasing the dose of the compounds you are using.
However, it is quite difficult to exactly mimic culture conditions described in other papers. There are differences in chemicals, culture environment etc.
May be you can try primary culture of hippocampal neurons as described: Development of GABAergic Synapses in Cultured Hippocampal Neurons
J Comp Neurol. 2006 Apr 10; 495(5): 497–510.
Catherine Croft Swanwick, Namita R. Murthy, Zakaria Mtchedlishvili, Werner Sieghart, and Jaideep Kapur
Regards
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I want to mimic a chronic nutrient starved condition in cell culture, how can I induce that ?
what are the parameters that I should look for confirming a chronically starved cell ?
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There are two kinds of starvation: one is serum starvation, where your cells will slowly arrest their proliferation (block in G1) for absence of growth factors provided with FBS, they will remain vital for some time but, depending on the cell line, they will eventually start to die. You should be able to obtain a good starvation in 24-48 hrs with FBS free medium. Another kind of starvation is fasting, so glucose-starvation. That is more complex to carry on over time, since your cells will not have any energetic substrate to use, will suspend their metabolism, and start to die quicker as glucose reserves run out (they could also undergo autophagy to survive and take time, but not for long). Anyway, cell cycle arrest in G1, observable with flow cytometry is probably the best way to confirm effective starvation. I don't know if there is a way to get a chronic starvation in cells, for sure is possible in animals, but I have some doubts about the possibility to keep starved cells alive for more than 48-72hrs.
If this does not answer to your question, you could provide additional details about what you need to do.
Good Luck
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I'm hoping someone with experience working with P. falciparum in vitro can help me with this!
I'm starting in vitro work with P. falciparum, and focusing on culturing gametocytes. I'll be working with strain 3D7.
Through my reading I've found that 3D7 is prone to losing it's gametocyte-formation ability when kept in culture for too long. A lot of papers suggest keeping cultures at a low passage number and continually thawing new feeder stocks to circumvent the problem.
My question is, what is considered a 'low' passage number? None of the papers I've read has given an actual number.
Thank you in advance for any help!
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Hi Danielle,
I used this protocol and it worked very well for me.
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I froze human PBMCs in 90% RPMI/10% FCS and 10% DMSO, but I saw some protocols that use neat FCS in the freezing medium. Should I repeat or would the current medium be sufficient to retrieve the cells back?
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It also depends on how you thaw them!
Into warm medium, work quick, consider adding DNAase which always improves!
Especially if you need many cells and viable!
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I am growing SW620 cells in L-15 media supplemented with 10% FBS and pen strep. However 1ml of a fully confluent plate subcultured takes over a week to become fully confluent. Any reason why this is so slow? The population doubling time is supposed to be 26 hours but this does not appear to be the case for my cells.
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Why are my SW620 cells even growing at cold 4 degree temperature? M using this to be a negative control.
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Dear all:
I'd like to treat our MSC with deferoxamine mesylate, DFO (Sigma), so that HIF-a is stablized. After reading its infomation sheet, I'm still clue-less on how to handle it.
I'm planning to prepare deferoxamine mesylate (DFO) at 100mM, as 1000X stock in water, and add 1 microliter to each 1 milileter culture medium. I'll bring DFO to room temperature 1 hour prior to opening it.
According to its infomation sheet, DFO will "decompose on exposure to air", suggesting me dissolve all DFO at once. However with my schedule of experiments, only a small amount of DFO stock is needed at at time, yet accoring to info sheet again, "solutions deteriorate on storage and should be prepared immediately prior to use" tells me not to use up DFO at once.
Would you kindly share your experience with me, please? I really really appreciate it!
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It's been long, and no one else answered, please excuse me for talking to myself.
I ended up dissolving DFO in ddH2O, and put it through 0.22um syringe filter to make it sterile.
The 100mM stock was aliquoted in black micro tubes and sealed with parafilm, frozen in -20C frig. To avoid repeated freeze-thaw cycles, I aliquoted it in very small volume, so that I can use it up within 2nd thawing. In this way, it worked just fine.
Hope this information could be useful to someone :)
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My undergrad thesis is all about contamination of water buffalo oocytes in the in vitro culture. Now, i've been researching for a possible antimycotic which I can use as a treatment. However, I've found Amphotericin B but it's too expensive, now I'm considering Ketoconazole as an alternative.
Does anyone tried using this? And may I know the concentration of it? Or could you suggest any other antimycotic which are not expensive?
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Unless you find multiple studies that made use of ketoconazole as antifungal for cell culture treatments, I advise you not to use it. Ketoconazole has been documented to antagonize GRs (glucocorticoid receptors) and if your aim is to maintain the integrity of the cells after an antifungal treatment, your drug of choice might not be appropriate.
Check if this supplement might be of lower price that Amphotericin: http://www.invivogen.com/fungin
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I'm trying to isolate rotavirus in Egypt
i need 0.5g of crystalline trypsin 
anyone have this in Egypt or comming to egypt soon
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I think that no one will help in this, the whole package is one gram and it is more than expensive. You have to buy or to share with others and also buy. All the best in your work.
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I have obtained result from in vitro which 10 ug/mL. How to predict what value of doses we need to use in the mouse (mg/kg). Is there any specific calculation to extrapolate the data to mg/kg? or we need to test the doses beforehand?
Your kind advice is greatly appreciated.
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Dear,
There is no such formula to extrapolate an in vitro dose to in vivo studies. But you can opt to any of the following
1. If your molecule is completely new, then go for LD50 studies first to estimate the maximum dose which is safe to use. Then select any three doses below LD50 (I recommend you to go below LD25) and proceed for your pharmacological studies.
2. If you know the chemical class of the drug and its therapeutic area, then you can refer to any reported drug molecule in the same class for its dose and route of administration.
3. If you have synthesized new molecules by changing functional groups in any existing molecule (Refer point 2)
4. You can go for Pharmacokinetic and toxicity studies but it will consume lot of time. Again, if you know the chemical class of the molecule you can at least get an idea of the kinetics and toxicities of your molecule.
Hope this will be helpful. have a great day.
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Dear all,
I was wondering how people add AraC to primary neurons in cell culture. I do a pre-dilution in H2O to achieve a concetration of 250 µM and add 5.6 µL of this solution to 500 µL medium in a well in a 24-well plate to get a final concentration of 2.8 µM at DIV 3-4. However, I was wondering if there are other protocols.
Do you use a higher volume? Do you exchange a part of the medium and add fresh medium together with AraC?
Unfortunately, most protocols only say "addition of AraC" but not the exact procedure.
Thx for your help!
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Dear all,
I now use the following protocol:
I take out 100 µL medium from each well (neurons are cultured in 24-well with 500 µL medium) and use this to make a pre-dilution of 14 µM AraC. Then I pipette 100 µL from this solution back in each well. This is how I achieve a final concentration of 2.8 µM in the well (more or less because there might have been some evaporation). I add the AraC at DIV3. At DIV8 I change the medium.
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Hello, 
I've been working on MTT assays and using hek293t cells. However, I have come across a problem. I seed my cells for 24 hours, then I add various compounds to the cells for 2 days, then I add my MTT dye. I've noticed when I add my MTT dye, I get a black precipitant that some what resembles a contamination. I do keep an eye on my cells for contamination within those 3 days of seeding and treatment, however, I do not see any signs of contamination. I only see this contamination/black precipitation AFTER I add the MTT dye. 
When I read my plates with the black precipitant I get inaccurate results, such as the reader will not give me a result, it will simply say "OVER". 
Has anyone come across this problem or have any idea what it could be? 
Thank you
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Dear Hanen,
Do you remove your test compounds from the cells before adding the MTT reagent?
Some compounds can react with the MTT and thus give false signals...
So, I would recommend to change the medium after the incubation time with the compounds and add the MTT in fresh medium or buffer to avoid a reaction with the test compounds...
Good look
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I will work on Parkinson's disease using SHSY5Y cell line. I have ordered the cell line from NCCS, Pune multiple times. Unfortunately, I have received the cell line contaminated with a slow growing antibiotics resistance bacteria. I have already checked the cell line from ATCC, but it will cost a lot of money around 70-80 thousands which is a big challenge for me to buy. So, I can't order it from ATCC. Please, help me. How to get the cell line? 
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Thank you Chunlin Huang sir!
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I have incubated several cell lineages (HEK, MEF, Hela) in a condition (DMEM high glucose, with amino acids but without serum - 1, 3, or 24 hr) for turning off mTORC1.
However, on this time-course of cell starvation, I have observed a downregulation of ~20-40% in p-S6-(Ser235/236) phosphorylation.
In my opinion, this effect is too modest for testing the effect of my treatment, even that my treatment completely re-establish the p-S6 phosphorylation levels in starved cells.
For every kind of experimental procedure, I always wash the cells with PBS before adding DMEM+FBS 10% (control) or DMEM without serum or DMEM without serum+my treatment.
Does anyone know the reason of this modest downregulation on p-S6? or Does anyone know a serum free protocol (except HBSS medium or medium poor of nutrients and vitamins) for turning off mTORC1?
Thanks a lot
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Dear Fu Jiayao,
Many thanks for your answer.
I have made 3 days ago an overnight incubation in RPMI (#1640) without serum and glucose, but with amino acids, etc. In sum, the Hela cells in starved condition detached from the culture dish, at least in my hands, therefore, I am not sure if they would survive a longer period.
I have made an overnight incubation with Hela and MEF cells in DMEM low glucose and with amino acids last night, and the cells were fine. I think until Saturday I will have an idea if this protocol worked.
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I'm using a protocol for in vitro gastrointestinal digestion comes a protocol already published with some modifications. Previous work does not explain how validated their technique. I read most of the articles that say they do validate their techniques by comparing the in vitro data with in vivo data, but in my case it is not possible to develop human studies.
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See the following review paper: The Combined Application of the Caco-2 Cell Bioassay Coupled with In Vivo (Gallus gallus) Feeding Trial Represents an Effective Approach to Predicting Fe Bioavailability in Humans. Link:
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I'm preparing an experiment in which I seed melanoma cells in collagen 1 gels. The stock solution of rat tail collagen 1 I am using is currently at a concentration of 10 mg/ml, and I am wondering if it would be possible to dillute this down to 5 mg/ml. If anyone has advice or experience doing something similar, I would appreciate the help. Thanks.
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You can dilute the collagen during the gel preparation procedure; often the collagen stock solution comes with a protocol for this where you plug in the desired final gel concentration and it gives the respective amounts of sodium hydroxide, water, and PBS you should add prior to incubation. 
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Hi good people,
I have got frozen vials of cell lines. I need to know how should I start with this vial for protein extraction? The thawing temperature, the centrifugation speed and all??
Kindly help.
Sumera
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 What Charles said. In your case I would first try thawing the frozen cells on ice (it will take a long time but since you don't care about long term cell viability it shouldn't be a problem) wash (add and then centrifuge 0C-4C 10 minutes 500-1000 rpm) the cells with ice cold serum free cell culture media once (maybe let them sit in this for a bit after first centrifugation)  then at least 3 times with ice-cold PBS to remove the dead cells, freezing media, and regular media components. For extra protection against degradation I recommend the Cell signaling technology lysis buffer with roche complete protease and phosphatase inhibitors for your cell lysis buffer cocktail. 
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I have isolated mouse primary myoblasts and have carried out a differentiation time course, lysing cells after 24h, 48h, 72h, and 120h of differentiation in 5% HS. By 72h, myotubes begin to form and by 120h most of the myoblasts have fused. The problem is that when I run these samples on WB, the tubulin normalization is horrible and very uneven from sample to sample. I quantify with Bradford assay and load 40 ug for all samples. I have done this routinely with all my other cell lines and get a nice even tubulin band across samples, but when it comes to differentiated myoblasts, it seems the quantification is not accurate. Any ideas as to why I am getting this unevenness, and what else I can try?
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Thanks Robert, that was very helpful! We have since checked in-house expression data for a-tubulin and found that our data corroborates the reference you cited - tuba1a (a-tubulin) expression fluctuates throughout myoblast differentiation. We will give GAPDH a try and move onto DJ-1 if needed.
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It is not possible to pipette a specific amount of the pure acetaldehyde. 
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Hi Ali,
In order to accept the impossibility of pipetting of the pure acetaldehyde, please look thru specifications of acetaldehyde, which has a density of  0.7850g/mL, packaging in AcroSeal™ Glass Bottle in Tin Can, and containing water 0.02% Max.
Best wishes,
Ilya 
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Hi,
I'm wondering what passage number is acceptable for MDA-MB-231 cells,
Some labs kill these cells when they are 20-25 passages.
Others Suggest it should be below 60 passages
Thanks
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Thanx alot for your help
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In our lab, I found many old stocks of the trypsin-EDTA solution, which was manufactured 8 years before. it had been stored in 4°C for these many years. Is it advisable to use that for our cell culture purposes? Will it have any impact on the growth of cells (Mostly for Hela cells and mouse macrophage cell lines)?
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Hi, I think that 8 year of storage are probably too much for every enzyme, also for trypsin that is a quite "highlander" enzyme.
I don't recomend to use it for your cells.
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I'm preparing a pilot to evaluate ideal time course and concentration to induce oxidative stress in cell lines (NRK-49F, B16, J774) with H2O2. 
For how long and in what concentration would you suggest me to do it? I'm thinking 1h, 2h, 4h and 8h and 1uM, 5uM, 10uM, 50uM and 100uM.
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Dear Natalia,
You should consider the state of H2O2. Is the most important thing, before performing the concentrations and times. best regards
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I have my drug compound which is not cell culture tested. Most importantly the drug amount is very less. How can I use the drug for cell culture?
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may be you should be doing a control test like seeding cells in a 60 mm or 30 mm dish and treat with very low concentration of your drug to visually check whether it is cell compatible or not. Before that you have to ensure in which solvent your drug is soluble. Then you can go for basic cell functional assay. Use the concentrations if reported or nearby concentration with different time points to check its IC50 on your interest of cell line
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We are facing the problem of contamination despite of using vacuumed tubes, even antibiotics and anti fungal drugs were also used in the media (RPMI1640 supplemented with 10% FBS .
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thanks Allende, we are using penicillin G and Streptomycin sulphate
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Hello, to continue with a protocol I need to do some optimization for a 96 well plate. One of the tests I need to do is have cells be at 60% and 80% confluency and judge which is better for transfection. However, I'm struggling to transfer the correct amount of cells and media. I'll go ahead and write out how far I've gotten:
A 96 well plate is confluent at 4x10^4 HeLa cells with a 0.32 cm2 SA of growth. So, if I want to seed the wells to have 60% and 80% the next day, I should plate 30% and 40% worth of cells due to HeLa doubling ~23/24 hours. Doing the math that comes out to 12,000 cells and 16,000 cells needing to be plated. I've counted my 100mm plate and found that (in this example) there are 2.4 x 10^6 cells in 10 mL within my plate. Where do I go from here knowing there is 100-200 ul of working volume for 96 well plates?
I'd like to plate 5 wells of 60% confluency and 5 wells of 80% confluency, but I'm not positive on how much volume of my 100mm cells I should add and how much fresh media to add. Ideally this would need to be a consistent process so I can repeat it for when I truly start my experiment.
Sorry for the long question, but I really need some help with this.
Thanks,
Adam
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Ok, so you want to seed 5 wells at each confluence. Let's calculate for 7, then. Generally, prepare your suspensions in a tube off of the plate first, then seed the resuspension onto the plate at the final dilution. (Not cells first, then medium).
Ok, so 7 wells at 200 uL per well will be 1.4 mL. This will be the final volume of your suspension before seeding. You can do this in a 2 mL tube with comfortable space.
7 wells at 12,000 cells per well is 84,000 cells. This is the number of cells from your concentrated original stock to the 2 mL tube.
You have 2,400,000 cells in 10 mL, which comes out to 240,000 cells per mL. By simple division this is (84,000 cells) / (240,000 cells/mL) = 0.350. The units cancel to mL, or 0.35 mL, which is 350 uL.
So, to get 84,000 cells, which is the number of cells needed if you evenly distribute your suspension across 7 wells for a final of 12,000 cells per well you need to dilute up 350 uL of cells from your original stock.
We know you need 1.4 mL of final suspension, so 1.4 mL - 350 uL = 1050 uL of medium to adjust the volume.
Take a 2 mL tube:
  • Add 350 uL of cells
  • Add 1050 uL of medium
  • Mix will
  • Dispense 200 uL per well into a 96-well plate for 12,000 cellls/well
  • You will have excess, discard or re-seed into a flask to conserve
Rinse and repeat for all concentrations you want.
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I was testing some extracts with this assay and all of them gave a % of viability higher than 100%. I´d like to know if there is anything I did wrong.
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Hi, from my little experience working with MTT, I have observed that sometimes over-confluence of the well plate could account for  your observation. Also ensure that you are seeding the same number of cells per well in the treatment group as you have in the control. Then some  literature search on your extracts and the particular cell line you are using could also give you a clue. All the best.
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I prepare 2mg/mL MTT solution in PBS, however, some cells metabolize and release from the bottom of the plate.
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Dear Edigar,
You are observing nothing unusual. Actually live cells via their enzymatic activity are able to reduce yellow MTT to blue formasan salts while dead cells can not. This is the basis of using this salt for measuring cell viability. I would recommend to use final concentration of MTT in plate at 0.5 mg/ml dissolved in phenol free incomplete media and filtered with 0.22 um pore size membrane.
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I need to study the SOD level in granulosa cells of the cumulus-oocyte complex. Can any experts tell me how to lyse the cells and count for 5 million cells/vial?
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Dear Isarin Thanaboonyawat,
First of all you should collect your cells in a 10 ml PBS after trypsinization or scrapping and then centrifuge to pellet down the cells. Before centrifugation mix cells well and count the cells using hemocytometer. Now carefully remove PBS without disturbing cells. Now you can lyse your cells by adding 0.2 or 0.3 ml pure water (may contain 0.1% triton 100X)  and again centrifuge at 10000 rpm and use supernatant for assaying SOD or catalase or protein estimation. 
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I am working on the cell culture system of A.testudineus. I have to adjust the osmolarity of PBS and medium according to the Serum of the fish. Please suggest me the right procedure.
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Dear Gayatri Rajenda Kachh,
You can use fish cell culture media available from Sigma or many other manufacturer. For cell analytical purposes, you can use PBS, HBSS where cell intactness is required or water if cell lysis required.
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hello , can anyone guide me on how many cells should i seed in one well (12 well plate) to have single layer of cells, and that would be best for transfection (siRNA). i have to start from primary culture, i'll use cardiomyocytes.any guidance, tips and suggestions are valued.
last time when i tried the cell density was too high but still i continued  and pcr results showed that transfection didn't occur. 
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@all ....many thanks for your guidance.
can i do transfection twice??  like usually we incubate for 48hrs after transfection, if i transfect it again after 24 hrs then would it affect the results? like it would improve the gene knock down or not?
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Is it possible to co-culture 2 different cell lines that require different growth medium (DMEM, and RPMI-1640)? Eg plating the cells on opposite layer (bilayer) of the tissue scaffold. 
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Hello Nur,
The problem you are dealing with is most common during co-culture experiments. Since tissue scaffold's one side can't be isolated from other so using different media for separate sides is practically impossible.  Ideally you should acclimatize your both cell types to single media which is totally possible or you should try a different media like a-MEM in which both cells show optimum growth. Still if your cells are very specific to grow in these two media then you can try RAFT MEM maintainance media from Lonza. That is suitable to culture most of the cells for co-culture experiments. Rest you can ask the company people.
Disclaimer: I am not intended to promote any company product and the answer provided here is solely based on my personal experience.
Hope it helps!
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I am performing a clonogenic assay after irradiating the 6 well-plates. I am finding that the middle 2 wells grow much more than the outer 4 wells, even when more cells are seeded in those wells. This does not have to do with the radiation exposure because even the control plate does this.
 I am assuming it is a humidity issue for the same reason that you don't want to use wells on the border of a 96 well-plate. How do I fix this for a 6 well-plate? Thank you.
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This is known as the edge effect and it happend in any tyoe of plate. It is related with  with different temperatures between peripheral wells and  central wells and with the evaporation that the peripheral  wells suffer. One solution that  helps is to preheat the plates for 15-20 mins in the incubator before seeding the cells, these helps to maintain an homogeneous temperature along the wells. As you mention for 96-well plates most people avoid peripheral wells  and fill them with PBS or medium to maintain humidity and an homogenous temperature along the other wells. If you need to have  6  experimental wells I suggest you to use 24-well plates instead of 6-well plates, preheat the the plates in the incubator before seeding the cells, and fill the peripheral wells with PBS (same volume that you uses of medium and cells) and use the remaining 8 central wells for your experiments. I hope you find this information useful 
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I know D3 falls apart pretty quickly but which one is it less likely to fall apart in during my exposures.
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DMSO
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Hi,
I'm trying to do an MTT assay with differentiated SH-SY5Y cells after compound addition. The cells were differentiated via sequential RA and BDNF addition to the medium. This all worked perfectly and in the end I have differentiated cells and the medium is DMEM with 50 ng/ml BDNF without RA and also no FBS.
For my assay I need to incubate the cells with different compounds for 24h. Do I need to still have BDNF in the medium or should I get rid of it and perform the assay in DMEM only?
Thanks in advance!
-Filip 
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Hi Filip,
In my lab, SH-SY5Y cells are differentiated by dibutyryl cAMP and RA for 48h. After that, those cells have pretty stable neuronal phenotype expression. So, we culture them for next even 48 h without differentiating media.
For me, yours protocol seems to be fine. So, go with that and Good luck!
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Briefly, lung, liver and spleen of mice was aseptically removed from euthanized animals. The tissue from  these organs was homogenized manually to homogeneity by using 1X PBS. Then, these homogenized tissue were serially diluted and spread onto 7H10 agar plates with appropriate antibiotics. But no colonies were observed. Could anyone help me with the protocol to be used for cfu counting.
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hi Adam Yes the M smegmatis used in vivo is resistant to the antibiotics used in the plates. As I have transformed it with the vector bearing antibiotic resistance.
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Does anyone know any side effect of using this inhibitor in cell culture? 
I've been using this inhibitor on melanocytes but quite frequently I have observed very weird effects of this inhibitor on the behaviour (the results dont match the expected prediction which Im very confident with) of the cells. Do you know if it somehow affects the internal cytoskeleton of the cells? Or if this molecule can be taken up inside the cells if incubated for too long (overnight) and mock up the cell behaviour?  
Do you know if it somehow affects the internal cytoskeleton of the cells? Or if this molecule can be taken up inside the cells if incubated for too long (overnight) and mocks up the cell behaviour? Even when you wash away the inhibitor after overnight incubation, the outcome afterward is still very weird and misbehaving.
Anyone who has experience, please share.
Thank you very much   
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Follow up with the question, I did another experiment where I incubate the cells with this molecule but did NOT wash it away but directly fix and stain it with phalloidin. From what I saw, the cells don't have their characteristic actin rich dots in them anymore. Usually my cells have a lot of these dots, but these dots just disappear in these GM6001 treated cells. It's really weird. 
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I have been using BT2 (benzothiophene carboxylate) and even though literature suggests that it is highly soluble in dmso, it precipitates out once added in culture media at higher concentrations. What are the ways to get rid of this problem?
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You're going in the wrong direction.  Increasing the concentration of compound in DMSO means using less volume in your cell culture - this DECREASES the amount of DMSO around, and thus minimizes the solubility of the molecule.  Similarly, filtering is simply removing compound from your solution, thus reducing the concentration beyond what you calculate.
What you need to do is figure out the LARGEST amount of DMSO your cells will tolerate, and then use the most DILUTE stock solution you can to supplement your cell culture media.
As for adjusting the pH: it might make it more soluble in DMSO, but you're adding it to culture media, which should be well buffered, and the little bit of acid or base in your DMSO should effectively cease to matter.  And that is, again, your problem: your compound isn't soluble in culture media.  Optimizing solubility in the DMSO stock isn't what you need to worry about.
As a further note: these compounds have been used in the literature a fair bit, on cell cultures.  You might just look at what other researchers have done.  And if that fails, why not just make the sodium salt of your compound?  Then you might not even need DMSO, as the salt should be relatively water soluble all on its own.
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I would like to ask whether should I change the A549 to grow in RPMI (and vice versa) for the coculture between A549 (which is growth in DMEM) and Raji ( which is growth in RPMI) for 12 hours or not. Will the different type of media interfere the characteristic of another cell line in coculturing? i tried to adjust the A549 to grow in RPMI by gradually add more percent of RPMI to the culture media. But at the 100% RPMI, it seemed that the A549 cell got stress and the stress maintained for the later passages :(
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You must check the amount of sodium pyruvate in RMPI medium, because A549 cells are very sensitive to the low level of it. If there is less sodium pyruvate in RPMI than in the DMEM medium, add the missing amount of that compund.
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Is it okay to cell stock without FBS?
usually I was using FBS 9 : DMSO 1 for cell stock.
But now I learn cell stock without FBS.
Here was just use 10% DMEM media 95 : DMSO 5
My cell line is breast cancer cell line.
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Hi Sena,
I always use Media: FBS: DMSO as 50: 40: 10 proportion and it works for every cell lines I have used.
Cheers
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Actually i am facing a problem that when i used drug about 1ug/ml concentration on MCF-7 and MDA, it reduces almost 70-80% population. But remaining 20-30% cells took almost 20 days to increase cell number upto 50-60% may be. during these days I have changed media for every 2 days with same drug concentration. I didn't transfer cells to the next flask because cell number was very low. but after 20 days cells got contaminated (Bacterial and Fungal) for being so long in a same flask. So what would you suggest, for that problem ??? 
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Thank you so much Alan D Brooks for your detailed answer. It helps me alot
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I am trying a co culture system where the cancer cells are placed in the insert.  However after incubation, I find the cell number decreased in control group however the ones in with the other cell did not.  I am using Millipore 0.4um PET.
Thank You 
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Hi Shilpa. I use inserts (Corning, polycarbonate and PET) to grow my cells - grows fine in both. There are various reasons for your situation:
(1) Have you checked if your cells need some coating material on the inserts? Eg. collagen, matrigel, fibronectin? 
(2) How many control inserts did you perform? If more than one, did all the replicates also show the cells dying on the inserts? If just one insert, try again one more time to see if it happens again. The cells probably just decided to die on your first experiment. My supervisor always tell me to do TLC (Tender Loving Care) on my cells. 
All the best!
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I'm working on a wound healing project and currently trying to see whether my extract has positive effect on cell proliferation. The two cell lines that I'm using are HaCaT keratinocyte and 3T3 fibroblast. After performing the experiment for several times, I found significant positive effect on HaCaT but not 3T3. Is there any explanation for this?
My method is as follow:
1) Seed about 5,000 cells per well for HaCaT and 3,000 cells per well for 3T3
2) Incubate for 24 hours
3) Serum starve with media containing 0.1% FBS for 2 hours
4) Remove media and add new media containing 1% FBS with extracts
5) Incubate for another 24 hours
6) Perform MTT cell viability assay
By the way, I'm using epithelial growth factor (EGF) as positive control and both cell lines show positive cell proliferation effect.
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Extract only works on some cells, but not all. Just like some cytotoxicity assay, some of the plant extract can suppress the growth of certain cell lines, but not all, because the mechanism inside the cells are different. 
Or you can try not using FBS when u want to incubate the cells with the extract, starvation allows the cells uptake more extracts. :) 
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Am beginner in animal cell culture technique. whenever i am trying to sub-culture the vero cell, often ends up in  granulation and vacuolation, finally arrest in cell growth. all the media and serum are kept in right temperatures. The antibiotic dose also up to the limit with right amount of temperature and also the CO2. kindly help me to find the  stress causing agents  which leads to  granulation and vacuolation.
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Thank you for your kind reply Maria
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We are doing potency assays on NUNC 439454 plates and we have been observing high background in a few wells randomly, while doing  these assys, even in wells where the dose concentration is less, we have changed the lots of plates but it is still  showing the same phenomenon. Is this problem with primary or secondary Abs used? Any help will be appreciated?
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In my experience the likely problem for spot background is almost always with washing errors, especially the last step prior adding substrate. Depending on your method of washing, you could try to increase washing volume and times (5x vs 3x). In larger screens I usually opted to leave the very last washing step for longer period.
As others pointed out, blocking is key, and the more important if you shake your plates.
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I'm culturing primary mouse astrocytes with mutant p53 in DMEM/F12 + 10% FBS. After a few days in culture, the media starts to rapidly turn yellow and the cells die.  The cells are mycoplasma negative, roughly 60-80% confluent, the media is clear (no obvious contamination) and the incubator is fine (37C and 5% CO2). Any idea what the problem could be? I've been testing my cells with the mycoplasma test kit from ATCC which is supposed to be one of the most sensitive and I'm the only one in the lab with this issue. 
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Are you able to culture other types, e.g. cell lines?  I wonder if perhaps the primary astrocytes are very sensitive to atmospheric oxygen concentrations (about 21%).  I suspect that they experience a much lower O2 partial pressure in vivo (about 3-5% perhaps) and possibly they can't keep up with the oxidative stress that atmospheric O2 puts on them.  I culture primary cells as well, and find that if I put them in 21% O2, the media changes to yellow almost immediately (no metabolites are produced that quickly) and the cells grow, just not very well.  Culturing in 3-5% O2 gets rid of that problem in my experience.  Perhaps you have a tri-gas incubator set up in your lab already -- if not, Airgas does offer 5% O2, 5% CO2, 90% N2 mixes for about $140 if I recall correctly.  You'd have to hook that up to an airtight container within your incubator.  But if it makes your cells grow well, it'd be worth it.
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In culturing L6 cells, using DMEM containing glucose with 10 % FBS. The cells would be seeded into 96 well plates and left to confluence and these would then be differentiated in DMEM with 2 % FBS for 5 days. Before tests, the medium will be replaced by RPMI1640 (2 g/L glucose) supplemented with 0.2% BSA. The medium will be removed after 2 h. My questions are:
1) How to source for this particular RPMI 1640 with this glucose strenght.
2) Hpw do I supplement the medium with 0.2 % BSA (How am I to prepare this?)
3) What should I use to remove the medium after 2 h?
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Dear Lakunle,
1) 2g/L glucose should be kind of standard formulation. If not, get lower strength and add your own, then filter sterilize.
2) 0.2% BSA means 2g/L. Proceed as above. Only makes sense when there is no serum present, of course (which will bring lots of own BSA)
3) I'd use a small pipet tip attached to a vacuum source. If your cells can stand it, just flip the medium out like when you'd wash an ELISA plate. If it is a short term experiment, you may do this directly at the sink, as strong sterility is not required anymore.
And wash the plate once or twice with the new medium in order  to remove traces of the old one. If medium should be too expensive, use PBS, ideally with an appropriate concentration of glucose.
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How many days do i have to incubate the cells after treatment in 60 mm dishes? I seed 100, 200, 400 and 800 cells in the dishes.
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Hi
You can try the protocol published earlier using LoVo cells in clonogenic assay-
Best of luck
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Since I am following the method of Makkar et al. (2007) but the result is not consistent.
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The ability of the various trypsin inhibitors to prevent trypsin hydrolysis of benzoyl-L-arginine (BAPNA ) ethyl ester is measured spectrophotometrically 410 nm.
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Can't dissolve cholesterol in culture media as it is water based. Dissolving it in DMSO or Methyl-β-cyclodextrin wont give the desired concentration i.e: 240mg/dl (Hypercholesterolemia).
Almost the same goes for you LDL too.
Any suggestions?
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Thank you Dr. Ali Abdulmajid for the suggestion its a good book and will definitely be helpful in vivo study, but I want to know if there is any way for in-vitro model 
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I have been trying to do this ROS assay with SH-SY5Y cells but the readout is incredibly poor with little to no variation even when spiked with H2O2 if anyone has done this experiment with these cells I would appreciate a protocol!
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The protocol from cell bio mentioned if i use a clear black bottom plate lysing is not necessary. http://www.cellbiolabs.com/in-vitro-ros-rns-assay this is the kit that i am utilizing. 
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I followed a protocol where peritoneal macrophages were collected and plated into 24-well plates (250000 cells/well) and non-adherent cells removed after 2h. After 24h, the DNA complexes were added and luciferase activity measured after 24 h. I did not have any luciferase active, not also in positive control! Not sure if the problem was the protocol or the complexes itself! Should I culture the cells more than 24 h prior transfection? Or what should I do different?
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You are welcome.
Positive control... well yes if you want to assess your current method, but maybe good to consider the Viromer RED as your main taxi.
Anyway, I renew my proposal, you are very welcome whenever you want.
Good luck.
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Generally I use virus but sometimes you just want to test something quickly. I hate CaPhosphate and LF3000 doesn't get many cells.
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Hi Paul,
For cultured neurons I would always go with lentiviruses. Calcium Phosphate has never worked so good for me in any cell culture.
Best,
Milica
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I need to solve alternariol and altertoxin II (around 2-4 mg) for incubation in cell culture (THP1 cells). Are they both soluble in either cell culture media or at least ethanol + water (80:20) ? 
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Hi, 
My HCC1937, MDA-MB-231, and BT-549 cell lines have all shown signs of infection 24hrs after waking up from LN. 
The images attached are from the same flask of HCC1937s. 
Would anyone have suggestions as to what could be done to prevent this? I had used the same TC hood as a colleague straight after her, and my cells showed infection while hers did not. I therefore, don't think it is related to the TC room, nor the TC hood. 
Any suggestions?
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It seems like that
use filter tips and filtered stripettes
package primary cell cultures with plastic wrap, it allows free exchange of CO2, but blocks any contamination,  Avoid using the waterbath., sterifilter everything into autoclaved bottles.
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We use the BrdU ELISA Kit from Roche a lot & never had any problems with it. Somehow, now we observe crystal formation (dark violett to black crystals) in the last steps of the assay... we did not change something major, like other cells, media or supplements...
Did anybody else experience this & has a explanation?
Thanks in advance!
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Roche has excellent customer support center in Mannheim, many specialists speaking several languages, just call them during working hours, they can assist you in real-time with your application, troubleshoot. If there is a defective product lot, you can inquire replacement.
Tel verde 0800 80 66 80
Or write them an email with your problem at mannheim.csc@roche.com  
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Apoptosis study (JC1/Flow): For Human origin lymphocytes cell lines in vitro study what would be the optimum incubation time to study the effect of test chemicals. Is minimum 4hr incubation with test substance enough? Kindly suggest? Protocols are most welcome.
Regards,
Samrat
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It may depends upon the mechanism of action of the drugs tested and upon the parameter studied. For an oxidative shock for example, 2-3 hours may be sufficient to observe cell death, if you want to study an effect on cell metabolism you have to wait until 6-12 hours at least and sometimes 24h, if you follow an effect on cell proliferation and growth, you have to follow your cell number during 24-48  hours, to detect apoptosis, it may takes 24h to 48 hours. I guess that for each compound of interest you have to determine a dose response-curve and a time-course.
Sincerely your's
D Duval Ph.D. 
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I need to prepare a solution in PBS, which is then mixed with culture media (RPMI) to about 30% PBS. Do you know if this high percent of PBS, overnight, has a detrimental effect on cell viability? 
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Thank very much for your help. Do you know if there is a maximum percent of PBS in the media that would not interfere with cell viability? Thank you again to both of you. 
Best regards. 
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For my experiment, I am treating my cells (NSC-34) with high conc. (~500uM/50K cells) of drugs (Etoposide in DMSO / only DMSO). Now after IF, I observed this  type of extranuclear structures in DAPI channel (see image). I am not sure if those are mycoplasma or dying cells particles from treatment.
If you see closely, few cells on the top seems healthy and do not show those structures around them. (I am not sure if all cells suppose to show extranuclear structures in case of mycoplasma contamination) Also, the transfection efficiency was about 60-70% which I think should be lower in case of myco contaminated cells. But again, I saw similar structures in untreated cells (although in much less amount) which makes me doubt those as contamination. Suggestions with more insights in this regards would be highly appreciated. 
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@Marcela, I have done Mycoalert test (Kit from Lonza) for my cell line samples and media. It came out negative. Can I trust the test and proceed with my cells?  
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Hi 
Could you please help me with this instruction to reconstitute  recombinant TGF-b :( To ensure recovery , reconstitute   at 20ug/ml in sterile 4 mM HCl containing 1mg/ml Human or bovine serum. 
Thanks for your help :)
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Hi both 
Thanks for your comments the amount of TGF-b is  2ug. 
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