Cell Viability - Science topic

I am delivering propidium iodide (PI) to live cells through liposomes. I would like to incubate cells and liposomes over a period x of time. Say x is 12h. Delivery will be random and might be, in some cases, immediate. PI will thus be, in these cases, in these cells for 12h.

I would like to be able to image the PI inside the cell. However, PI is toxic to cells - how long can a cell last with (a certain concentration of?) PI, before it degrades and can no longer be imaged?

Thank you in advance for your help.

Hello. I am trying to test my designed peptide binding towards MDA-MB-231 and MCF-7 cell lines. However, I first need to dissolved my peptides as they are in lyophilized form.

The suggestion from my peptide synthesis service provider is to use 1 part ACN: 3 part water, which definitely is toxic to the cell lines. Alternatively, I can use DMSO as my solvent.

From my reading, concentration of >1% DMSO in my media would be cytotoxic to MDA-MB-231. I am now trying to run MTT assay to test the concentration that would be minimally cytotoxic to my cells. I am testing 1%, 0.5%, 0.25 %, 0.1% (v/v) DMSO in media.

However, I am curious does the concentration of this DMSO affect my peptide solubility in the solution? If let say I want to prepare 10uM concentration of my peptide, then if I am to prepare it by dissolving it in 100% DMSO, when i dilute the DMSO to 1%, wouldn't that also dilute my peptide? Or do I need to prepare higher than usual?

My concern is that my peptide sample is limited (10mg) per peptide so I don't want to use up whole sample as I have another assay to run.

Any advise on this? Thanks for the help!

Hello everyone,

I am currently working on a project involving primary cultures of breast cancer tissue samples, and I have a few questions regarding preservation:

How long can I safely preserve tissue samples at 4°C without compromising cell viability and functionality?

Are there specific preservation media or additives you recommend for maintaining tissue integrity during this period?

What methods or assays can I use to assess the viability of the cells after preservation?

I appreciate any insights or experiences you can share!

Thank you!

I am have purified an bioactive compound (drug) from a plant. I need to perform an MTT assay to check cell viability and to determine IC 50 value.

After lyophilizing of HPLC fraction the compound is not visible and also according to the HPLC report the concentration of the compound is very less.

What should be my approach?

When IC50 cannot be obtained according to the cell viability chart, what should be the dose selection under cell culture conditions? Would it be beneficial to go with sham control?

For example, for this chart 1) control vs. 0.3 mg/ml, or 2) 0.0025 mg/ml (sham) vs. 0.05mg/ml. Which one should be preferred?

What reference can I give to base this on literature and how should I explain it academically?

Thank you in advance for your help.

If I wanted to do cell viability using mts assay, the treatment need to be diluted with complete media or basal media? What happen if I used complete media to dilute the treatmemt and do cell viability test?

I intend to analyze some aspects of cellular immune response after vaccination on those who didn't seroconvert, comparing with a control group of people who seroconverted, and at different periods (including pre-vaccination). Since I can't tell who will or will not seroconvert, I'll have to collect and keep blood samples from everybody pre-vaccination and later select and analyze those who didn't seroconvert (which are the minority). Given that isolation of PBMC with a density gradient is a time consuming procedure and I'll have numerous samples, I wonder if I could just separate and freeze the buffy coat from all samples. Later on when I get the serology results, I could isolate PBMC from the thawed buffy coat with Ficoll only from selected samples. I know cell viability will probably be lower compared with that from fresh blood. Does anyone have this experience? Can anyone help me by giving some advice? Thanks in advance! 😊

I was performing an MTT assay to check cell viability. Due to a technical issue in the plate reader I was using, I had to read the plate at 540 nm wavelength. Would the results still be credible?

I need to perform an MTT assay on MDA-MB-231 cells to determine cell viability. I am using free docetaxel (DTX) as my positive control for my MTT assay and my supervisor has asked me to dissolve my DTX in DMSO. However, it has been specified that my vehicle for the positive control as well as the positive control itself cannot contain more than 0.1% DMSO otherwise it will be too cytotoxic and we won't know whether it is my treatments killing the cells, or the DMSO.

I need to work with concentrations 10, 20, 30, 40, 50, 60, 70, 80 and 100µg/mL of DTX to treat my cells with. Therefore, how do I create a stock as well as serial dilutions of my DTX in DMSO and DMEM media such that the final concentration of DMSO that I add in the wells is less than 0.1%?

Hi Everyone

First year PhD student in London here, I'm new to Caco-2 cells.

I have two issues that are bugging me. Does anyone have any advice on how I can navigate these problems?

The first one, as shown in the first image, where cells seem to clump together in a wall like structure, and there are cells growing on one side but none on the other side of this 'wall'. This is super annoying because I seed the cells and let them grow for a week, only to find that the cells on one side of the plate have died or disappeared so I essentially waste a week and media.

The second one, are the black rod like structures bacteria? I'm treating my cells with media in the absence of antibiotics for a gentamicin protection assay for 24 hours but normally, I do use pen-strep 1%. Cell viability in the potentially infected regions appears to be severely limited. However, the media is not cloudy?!

Thanks so much!

Pranaya

I am currently testing the effect of bacterial filtrates on cancer cells , after seeding the cells I tested the bacterial filtrates against them , and got images of all 96 wells using inverted microscope. How can I calculate the % of viability without staining the cells or doing MTT ?

i would appreciate any help

Hello, I am currently conducting research on ovarian tumour cells and the use of chemotherapies with dietary metabolites and their combinations to see if there is a difference in cell viability. I have conducted tumour cell assays and have formed bar graphs with the normalised data so the x axis is the therapy concentration e.g pemetrexed at 100nM, 1um, 10um and 100um and the y axis is cell viability (%). I am unsure on how to analyse this data, I have chosen ordinary one-way ANOVA as the independent groups could be the different treatments with different doses to see if there is a difference in cell viability. I have run the tests and they seemed to have work but I have no actual understanding of the results and I also used one sample t-test, which again has given me results but I am not too sure what is showing. I got told that it isn’t clear what comparisons I am making – a given observation must be significantly changed compared to something else – but what? Any advice on what statistical analysis I should be using?

Hello,

My E. coli cells express both green fluorescent protein as well as mCherry. So I need a fluorescent stain of color other than green and red fluorescence to enumerate their viability. Please suggest. Thanks in advance.

the best cell lines for myeloid leukaemia for transfection

Dear colleagues,

I need to transfect a 10 kb plasmid into Caski cells and am hoping to use Lipofectamine 3000 for the transfection. If anyone has experience using Lipofectamine 3000 specifically in Caski cells, I would greatly appreciate if you could share your transfection protocol. Specifically, tips on:

Ideal plasmid:Lipofectamine 3000 ratio

Optimal cell seeding density

Lipofectamine reagent dilution and complexation steps

I want to achieve high transfection efficiency while maintaining good cell viability. Please let me know the optimal conditions and procedures you’ve used for transfecting plasmids into Caski cells via Lipofectamine 3000. Thank you so much for taking the time to share your experience!

MTT assay

Why does a negative or a 0 for blank mean? Does this mean that cell viability has stopped?

I am working with a formulation of calcium phosphate bone cement reinforced with graphene, and I will perform an MTT assay to evaluate the cell viability of this material. However, I need to know which bone cell type is most commonly used for this assay. Thank you in advance.

Hello,

I am Mahmuda, now I am working with DG44 cell culture. So far, my cell culture viability improved to 90%. However, before transfection, my cell viability decreased to 70%. I am very disappointed with the results of this culture.

Is there any suggestion or input that can be given so that I can solve this problem?

Then, is there any particular trick to do for this DG44 culture?

Thank you for the help

I have WST-8 powder, how to use it to prepare CCK8 solution for cell viability testing

I am new to using MTT assay. My experiment is to treat cells and see the cell viability after 24,48,72 hrs so should I grow cells in three different plates and put MTT reagent for each one separately or there is a way to read every 24 hrs until I finish the experiment?

Hello everyone!

MTT was performed after applying DEHP to HeLa cells. According to MTT results (24 hours of exposure), cell viability is significantly reduced at concentrations of 100 micromolar and above. In the 1-30 micromolar range, viability is almost the same. Next, I will check SOD, CAT, GPX levels with ELISA. What concentrations should I use in ELISA? Should I include IC50, IC30 or IC20?

I would be glad if you help me.

Thanks in advance!

Our CO2 supply is outside in a cage and there is fairly long distance pipework going around the outside of the wall to the entry point in the wall of the lab. Since turning late Autumn/early winter, some of our cells are starting to look a bit odd. It seems to get worse the colder it gets. The incubators are reading 5% CO2 (so unlikely a leak), the temperature is correct too. They are newish incubators, only serviced recently (we are getting our own CO2 meter to check soon too). All reagents were replaced (several times). Two different people have had the same problem, so not user error either (both experienced users). Different batches of cells have been tried too. Its the first time Ive ever used a supply from outside, (its usually next to the incubator) so I was wondering if it had an effect on anything as Im running out of ideas. Many thanks

1. Take the vial with the cells and thaw it at 30 degrees in a water bath.

2. As soon as the culture is thawed and liquid, remove the vial from the water bath, and clean it using 70% ethanol. Take the 1 ml cell culture from the vial and add it to a 10 ml falcon containing 4 ml of complete Schneiders media (Complete S2 Media: 10% FBS, 1% Pen-Strep, 89% Schnieders Medium). Mix them nicely with pipetting.

3. Place the falcon, now with 5 ml components (1 ml of culture from stock and 4 ml fresh media) in a centrifuge at 100g for 10 min.

4. Remove the supernatant, which would contain DMSO, and then add fresh 5 ml of media (2.5 Fresh S2 Complete Media + 2.5 Conditioned S2 Media) and add this culture to the T-25 flask and let it incubate for 3-5 days before starting passaging.

Am I doing something wrong while I reculture the frozen stock?? Or is it alright and I should just clear out the dead cells using low centrifugation speed for 10 minutes or so?

I am combining drug A (IC20 and IC50) with drug B (5 uM, 10 uM and 50 uM). Drug B reduces cell viability to 28% at 50 uM, while drug A IC20 combined with drug B 50 uM reduces cell viability to 32%. CompuSyn gives a CI=0.46. Is this correct? To have synergism shouldn't the viability be reduced to less than 28% when combining the 2 drugs at these concentrations?

Thanks for any help.

Im evaluating cell viability in flow cytometry with propidium yordurate and I need to keep my cells alive in a solvent that can keep them up to 1 hr and can also be used to be injected in the flow cytometry.

Hi,

I am curious to know what is the acceptable cytotoxicity levels for a drug/compound tested using MTT assay in vitro?

Hi guys!

Lentiviral CRISPR-Cas9 targeting guide RNA (gRNA) expressing vector is the most common method to generate a specific gene knockout cell line. However, during my recent research work, I found the RAW264.7 cell line quite difficult to be transduced by Lentivirus. Meanwhile, after transduction, RAW264.7 gets activated and polarized with poorer cell viability and slower proliferative rate, in which case it becomes tough to conduct the further study on it. As in the same situation for immortalized bone marrow-derived macrophages (iBMDMs). In the methods of some classic reseaches about RAW264.7 or iBMDMs, most researchers utilize Lentiviral transdution to fulfill genome editing. How can they avoid the abovementioned problems(poorer cell viability and slower proliferative rate after lentivirus transduction)?

I'm pretty new to this, so any help, tips, advice, or direction would be very helpful. Thank you in advance for any suggestions.

If we treat NHDF cells with ascobic acid for 1 hour at 33ug/mL and then irradiate the cells with a low dose of UVA we see a good antioxidant response and cell viability does not change compared to non-irradiated NHDF cells. However, if we followed this procedure but with a incubation of ascorbic acid for 24 hours, after irradiation we were unable to detect an antioxidant effect and we also observed an increase in cell viability.

We know that ascorbic acid can reduce our cell viability reagent, but we do not know why its antioxidant effect and viability depend on its incubation time. Do you have any idea?

Hi everyone,

We are recently doing cell viability and for that we use Alamar Blue. The results after ascorbic acid treatment and UVA-irradiation show increase cell viability for cells that were treated with acid ascorbic. We think that there is no change of cell viability, that this increasing is due to increse in metabolic activity of those cells. Could it be possible? Do you have experience with a treatment that increse metabolic activity? There is some way to only see cell viability with Alamar Blue?

Thanks in advance,

I have a compound (C23N3OH27) to repeat some results with a molecular weight of 361.48. The problem is that the results are not being the same, I am evaluating cell viability (K562 and KG1) with resazurin (24 hours of plating 20.000 cells/100uL, 24 hours of treatment 100uL, 4 hours of resazurin 20uL) and the results lead us to believe that it does not induce death in any of the cases. concentrations tested (30 uM, 20uM, 10uM, 5uM, 1uM), I have already evaluated cellular metabolism, resazurin, interaction of the compound with resazurin and none explains the reason for not repeating the results. I am suspicious that it could be my dilution, I used a table from a colleague that performs the calculation automatically. Could someone help me to do the dilution directly just so I can assess if it's correct? I have 5g powder of the compound which was diluted in 2305.34uL of 100% DMSO, which according to the table gave me a solution of 6,000uM, I don't know if that's correct.

Thanks for all contributions!

I have attached the dilution table below.

I am writing to report a concerning issue I've encountered during my recent migration assay using RAW 264.7 cells. I have followed the standard protocol, but I am facing a serious challenge with cell viability and scratch healing in response to CXCL9 treatment.

Here is a detailed description of the experiment:

Cell Line: RAW 264.7 cells

Media: DMEM Free Serum (used to standardize the results, Raw cells 264.7 grow in DMEM with 10%)

Cells were plated in 12-well plates until they reached confluence.

Media was removed, and cells were washed with PBS.

A scratch was made using a 200μl tip.

Cells were washed again with PBS.

Fresh DMEM Free Serum media containing varying doses of CXCL9 cytokines was added.

The issue I'm facing is that after the addition of CXCL9, the scratch disappears quickly and cells seem to detach and float, indicating a loss of cell viability.

I would like to inquire whether I am following the correct protocol, if a reduced FBS media is preferable over serum-free media, or if you have any alternative suggestions for conducting the migration assay with RAW cells.

Usually neuroblastoma have low transfection efficacy (5-10%) when transfected with Lipofectamine 3000. So, this time I have tried electroporation with this condition,

poring pulse: 175V, 2.5ms, 50ms interval, 2 pulses, 10% decay rate, + polarity

transfer pulse: 20V, 50ms, 50ms interval, 5 pulses, 40% decay rate, +/- polarity

I have used this transfection condition with three seeding density (80K, 100K and 150K)in 24 well plate.

However, the cell viability is very low for RNA collection after 24 hour of electroporation.

Could anyone please suggest me how to improve the cell viability?

TIA.

Regards

Ayman

I have used the MTT Assay to measure cancer cells' viability under an antioxidant compound's influence. But contrary to expectations, with the increase of antioxidant concentration from 5 μM to 150 μM, the viability not only did not decrease but also increased. In other words, with the increase in the concentration, the amount of light absorption increased. In your opinion, what is the reason for this technical error? Or what kind of problems could have occurred during the MTT Assay?

Hello to all,

I am working on SH-SY5Y cells and I treated my cells with Rsl3 and ferostatin-1 (24 hours) but I could not see good cell viability. I would like to know how I can treat my cell so that fer-1 can prevent cell death caused by Rsl3.

With thanks for your attention

Parisa

I have done a number of MTT assay's for a study I am a part of, twice the results showed a very low viability and this crystal formation. I did a later assay following the same procedure, using the same cell line, and same MTT with viable results. What would cause this? Could the MTT mixed for that trial being exposed to light have anything to do with it?

I isolated and differentiated bone marrow-derived macrophages from mice. After differentiation I frozen them and kept them in liquid nitrogen. Then, I thaw them and let them recover for 3-5 days before polarization.

They proliferate perfectly with the differentiation medium (DMEM + 20% FBS + 30% L929 SN), and I can keep them in culture for, at least, 2 weeks after thawing. To induce polarization I split the cells, count and seed them at a density of 100-150,000 cells/cm² in classical medium (DMEM + 10% FBS). I culture them for 24h with this medium, after I change the medium to the one with the specific cytokines to induce anti- and pro-inflammatory polarization (IL-10 and protein homogenate from injured muscle, respectively) for 48h. After 72h from seeding, I get a viability of 10-15%, which is too low.

I have several questions for which I haven't found a clear answer yet.

I attach the photos from the BMDM after 24h of seeding with differentiation or classical medium.

Hope you can help me!

Thanks :)

I'm doing primary tumor spheroids to a drug screening. We're having success to make the spheroids but I'm not having succes to dissociate the spheroids to evaluate the cell viability (using trypan blue to count).

I tryed to use trypsin 5 and 10 min (37ºC) and Accutase 10 min (R.T.) and the spheroids not dissociated completely. Could you help me?

p.s.: Spheroids were plated with 5000 cells, so they are ver small to dissociate mecanically.

Thank you!

I checked the literature in detail to compare different cell viability, cytotoxicity studies to check the biocompatibility of biomaterials. In some articles, they use the 'leachable-conditioned medium' method to check the biocompatibility. For this purpose first, they incubate biomaterials in cell culture medium without FBS after that they use this 'conditioned medium' with FBS in cell seeding. I am wondering in this first step why they don't add FBS directly to cell culture media and is it important for this procedure. I also saw different methods including FBS.

I am waiting for your response. Thanks:))

I want to check the cytotoxicity of my material. I am using MTT assay kit. The protocol is provided in the booklet with kit how to perform the assay. Is it better to follow the protocol provided in the kit or use protocol given in literature review. As in literature review different protocols are given performed with different kits.

what can be the working conc of the dye in a 96-well plate experiment?

As you are aware, Acridine orange is an intercalating dye that can permeate both live and dead cells. AO will stain all nucleated cells to generate green fluorescence. Propidium iodide can only enter dead cells with poor membrane integrity, so it will stain all dead nucleated cells to generate red fluorescence.

So, it is basically straightforward to calculate the cell non-viability using the PI. However, the AO enters all cells regardless of alive or dead. So how can I analyze the raw data to get meaningful cell viability/non-viability percentages?

I have positive and negative controls, and blank wells as well.

Thank you in advance.

In literature 30mM glucose concentration cause reduction in cell viability on HK2 cells but we tried different high glucose concentration even for 48 hours but no big difference on cell viability. what could be the possible reason for this outcome. How to address this problem, did anyone faced similar kind of issue while studying type 2 diabetics using HK2 cells

Hi all, I have problem with my hepG2 cells.

So before covid lockdown, I froze my cells and put it in Mr Frosty in -80°C. I used Cryostor as the freezing media for 1st batch after I received the cells (since I was adviced that HepG2 cells viability is better with Cryostor). Though after that I replaced it with 10%DMSO in FBS due to the lack of Cryostor at that time. The numbers of the the cells I stocked varied between 3 to 3.8 X 10^6 cells per cryovial.

We transferred the tube to LN2 after about 3-4 weeks (we waited other cells to remove it to LN2 together).

And now we want to wake them up, but the HepG2 cells do not attach to the flask the next day after I thawed using standard thawing protocol (from ATCC). They become aggregates and are still floating in the media. Are they dead?

I tried with other tubes from different batches already, but the results are the same.

I wonder whether this is caused by the prolonged time in -80? The other cells are fine when we thaw them.

Or is this because of too many cells per vial? Though I saw other people froze 4 X 10^6 to 1 x 10^7 cells...

When silver nanoparticle used as test compound for cytotoxicity studies (MTT), the results showed like the compound is toxic upto a concentration, then onwards increased cell viability with increasing concentration of the drug. Is it possible?

Hello, I am working with cell culture and I need some advice. I usually use trypsin to detach the cells from the flask, but I wonder if I can stop the trypsin reaction with serum-free medium instead of serum-containing medium. Is this possible or will it affect the cell viability and growth? How do you subculture your cells with trypsin? Thank you for your help.

I have been having trouble culturing J774a.1 and at my wits end (I have posted a similar question before).

So my lab got a new vial of J774a.1 from ATCC. I thawed them in DMEM (from Hyclone). For splitting, I detach the cells using cell scrapper. The viability of the cells are at 50% for the first passage which is not surprising. However, the viability dropped to 30% subsequently, and then 20%. I changed the media to RPMI with HEPES and L-glutamine (from Hyclone) and the viability increased to 50% -60% but never more than that. Moreover, the cells only grow to 70% confluence at most. It could be my way of scrapping causing cell deaths though (I scrap as gently as I can).

So in my last ditched attempt, I seeded the cells into a non-tissue culture petri dish. They grew really well, with 90% viability. However, most of the cells are not attached (as expected). But when I seed them back into a TC flask/plates, they attach well, but again, do not grow beyond 70% confluence. Is this normal? I have yet to check for mycoplasma contamination yet though.

Any ideas how I should approach troubleshooting this issue? I am out of ideas. Please help!

Thank you so much!

I need to perform cytotoxicity studies with an isolated substance for a future formulation with oral administration. However, I don't know which cell lines to choose to test cell viability... Thanks in advance!

Let's assume that when the cell viability experiment was conducted using animal cells other than HeLa cells, the absorbance of the negative control (cells not treated with Doxorubicin) showed almost no difference from the value of the blank, and cell viability was not well measured. . In this case, please describe a strategy to solve the problem.

Hello everyone

I have a question concerning the use of the Alamar blue cell viability test for cells encapsulated in an alginate-based hydrogel. Does this test fit with the gel? Does the compound reduced by the cells get out of the gel into the culture medium?

Hello,

I am measuring multiple different parameters in cells using fluorescent channels in flow. I would also like to assess well viability. Given that I take the same volume from each well, and do not define a stopping event, can I compare live events between wells as a viability measure? I understand that not all cells that appear live in flow are viable, thus the use of viability stains. Do such stains have to be used to generate viability data from flow?

Thanks!

I am trying to distinguish the pillar cells from thin and thick epithelial cells of Chinese mitten crab so that I can study using pH dyes regulatory capabilities of the individual cell types. Cells can not be fixed as I am doing live cell imaging and I should avoid other live cell dyes which may hamper cell viability. Attached is a picture of an intact gill lamella.

Drug toxicity test against normal cells

Hi there,

I used L-NAME to treat cancer cells. Then I did LDH and MTT, according to these results, MTT showed the cell viability was above 100% compared with control, and LDH showed cytotoxicity was nearly unchaged.

However, I saw some studies suggested that L-NAME could inhibit cancer cells proliferation. I'm confused with it.

Does anyone have some experiences with it?

Many thanks.

Hi, I am performing MTT assay to measure cell viability of ovarian cancer cell line (A2780) upon treatment with Cisplatin. However, I am repeatedly getting the following results and not sure why I have values in negative.

Can someone suggest me a possible explanation?

I have done two tests to investigate the effect of a material to cell viability and how cytotoxic the material is.

Briefly, I seeded cells in a 96-well plate and introduce the cells with the media treated with the material, and then place them in the incubator for 24h before testing. The next day, I did the PrestoBlue test and followed by the LDH assay.

When I did the PrestoBlue, the control contains of huge number of cells which is making sense for me as it is a control. But when I did the LDH assay, the reading between control (media + cells only) is quite high compared to the blank (media only). From what I understand, this means that cells are dying in the media, but this result does not correspond with the PrestoBlue result.

Can anyone help me explain what is going on? Is it a user fault (my fault) or is there any scientific reason why the results are not corresponding to each other?

I treated my cell (HL60 and Mec-1) with Graviola leaf extract and Vitamin C alone and in combination form but the viability of cell increased in all treatment and the assay which I used was MTT. Can anyone know the reason behind increase cell viability and how to overcome this problem?

Aslamo Alikom (Greetings) Everyone,

I've MTT results for the cell viability after an exposure to an inhibitor alone and combining it with another drug.

Now I wanna see how the other drug affects the IC50 of the main inhibitor.

Cell viability% for the drug alone and with combination ranges from 100 to nearly 40s and 30, respectively.

I was wondering whether I've to normalize the data before doing non-linear regression to calculate the IC50 on Graphpad Prism.

I would highly appreciate your advice and guidance in this matter. Thanks in advance

actually, I want to calculate growth rate of my cho cells. I have its viable cell count, total cell count, and viability percentage. so is there any formula for growth rate calculation using these values?

I did a MTT assay for a plant extract against L929 cells. But in readings, the cell viability percentage was increased more than the untreated cells, with increasing concentration.

I have been working with a ceramic based scaffold, and it is to be determined for cell viability, for which I have chosen MTT assay. Although I have tried seeding cells onto the scaffold or taking extracts of the scaffold and then treating them to the cells, I was unable to get proper results. In general, the problem faced was that the absorbance of media control is remaining higher than that of cell control, which in general shouldn't happen. It would be great if any researcher can help me sort out the issue or suggest any other method that can be followed to perform a cell viability assay.

I treated cells with a toxic compound and was trying to find the most suitable treatment concentration. After performing a cell viability test, I saw that the middle concentration reduced the cell viability, but higher concentrations did not cause a reduction in cell viability. Does someone know what the reason could be?

I am facing difficulty in estimating cell quantification in my scaffold. Usually live/dead assay and MTT is performed to check cell viability. But what if we can extract cells directly from scaffold and measure it using a cell counter.

I am trying optimize 2L bioreactor process with SP2/0 cell line. This is the third batch which was initiated. The initial batch parameters are 0.5x10^6 cells/ml, 37 degree C, air and O2 cascade: 0.09 and 0.03 vvm. The viability of the cells used for inoculating the batch was between 90 to 92%. But i am observing one common trend during all batches. From first day onwards, though there is doubling of cells but the viability decreases as the days proceed. And from day 2 onwards the doubling ceases and viability reaches to 50%. By the end of third day i need the discard the batch.

I am unable to understand the situation. If any have ever observed this kind of situation while working with SP2/0 cell line. Please share their experiences.

I am working on a probiotic that have anaerobic bacteria spp. This probiotic is available in capsules commercially from Seeking Health LLC USA.

I need to dissolve the capsule powder in anaerobic PBS. In resulting Bacteria suspension I want to check the viability of the suspended bacteria.

I did a literature survey and found that few authors are telling to handle it dry anaerobic conditions. Mix the powder in anaerobic PBS and homogenize it. After this I can check cell viability via flow cytometry.

Do the homogenization maintain the cell integrity and viability?

If yes, what are the homogenization conditions to maintain the cellular integrity and cell viability.

Can I use Trypan Blue dye exclusion method for the viability check?

Or only Fluorescent dyes (Flow Cytometry) are the option ?

Expert advises are needed, please help me in this.

Thanks

Regards

Anuj

We want to get single cell suspension for flowcytometry staning from mice colon lamina propria.

We can only make about 30-40% cell viability after digestion.

We have tried different enzyme concentration(1000U and 2000U for collegenase VIII, 5mg and 10mg for collegenase IV in 10mL) ; dierent digestion time (1h and 2h);different wash time(10min twice; 20min once; 20min twice); But none of them is capabel to make single cell suspension over 70%.

Why?

We used followed protocol:

PBS wash tissue

↓

PBS + 30mM EDTA + 1mM DTT, 220rpm, 37°C 20min in Shaker-Incubator

↓

PBS + 1mM DTT, 220rpm, 37°C 20min in Shaker-Incubator

↓

Hand shaking for 1min

↓

wash tissue with PBS and shear the tissue to 3mm slice

↓

Digest with 100U/ml collegenase VIII and 150ug/ml DNase I for 1 hours

↓

Pass cell through 70um filter

↓

Stain cell with AO/PI to calculate cell viability.

Thanks so much for your suggestion.

My work aims to observe cell viability through AO/PI staining. Although I found numerous AO/PI staining protocols, but most of them analyzed the viability through microplate reader or cell counter (countess).

We're trying an approach of observing these cells under microscope with fluorescence setting. I plan to seed my cells into 96 well plates, and later stain them with AO/PI staining. However, I couldn't find any detailed protocol for this.

It seemed like AO/PI stain was diluted with PBS, prior staining - but this was for microplate & countess protocols.

Should I dilute my AO/PI stain? if yes, how much volume is needed? I'd also like to know if there is any incubation period before observing my stained cells under microscope.

Thank you!

I'm using the standardised INFOGEST - Minekus (2014) consensus paper for my simulated in vitro digestion model but each time I use the digesta (intact, diluted 1:4 and 1:8) on the caco-2 cells to assess carotenoid uptake and transport, the cell viability is less than 10% based on the MTT assay. I strongly suspect the bile salt concentration I'm using could be toxic to the cells. Is there a step by step procedure I can use without necessarily using the test kit to verify the actual concentration of my bile salts?

Hello There

I´ve been trying to do a sulforhodamine cell viability test for NSC-34 and it is not working. I changed the protocol to minimize cell detachment (washing only once, using collagen) but when I finish, there is almost no cell in the well. I see that some papers use LDH assay to cell viability in NSC-34. Is it better to change or am I letting something pass?