Cell Viability Assay

Cell Viability Assay

  • Daniel J. Medina added an answer:
    Has anyone tried altering the suggested dilutions given in invitrogen Click-iT EdU Imaging Kits?

    I have read that (as usual with kits!) the amount used can be significantly dialled down to make the kits go a little further. Does anyone have experience with this?

    Daniel J. Medina

    Hi Olivia,

    Yes, I just make the solutions more dilute and then use the recommended mix for the final cocktail and follow the remaining protocol

  • Nazli Alizadeh-Tabrizi added an answer:
    Can MTT assay be taken as equivalent to cell proliferation assay (BrdU)?

    Im currently working on SH-SY5Y cell line. The treated group showed increased BrdU +ve cells on Immunocytochemistry technique but % cell viability from MTT assay showed no significant effect. If there's increase in cell proliferation, % cell viability should also increase, at the same time period of treatment. I would really appreciate your help analyzing this case. Thank you. 

    Nazli Alizadeh-Tabrizi

    Please check the question and  related answers :

    Can % viability be considered an accurate representation for proliferation measure by MTT assay?

    Hope it would be useful.

  • Amanda Villalvilla added an answer:
    Does the optical density of formazan dissolved in DMSO change over 24 hours?
    For ease I would like to perform an MTT assay and dissolve the formazan in some of the wells of my plate while other wells continue to be treated at 37 degrees overnight. By the time I have added MTT to the final treatment condition and I solubilize the formazan, it will be close to 24 hours since I solubilized the first wells. Over the 24 hours, is it likely that the optical density will change significantly enough to prevent a fair comparison of the two conditions?
  • Hugh J Byrne added an answer:
    Which cell line is best for Solid Lipid Nanoparticles Skin cytotoxicity?

    I am plannnig to perform skin toxicity studies for my solid lipid nanoparticles for skin delivery. I am in a fix which cell line i should use? L929 mouse fibroblast cell or mouse 3T3 fibrobalsts cell...? 

    Hugh J Byrne

    Similar to Claudia, we also use HaCaT cells. Skin models are expensive, and be careful about their use for permeation studies.

    “Comparison of Structure and organization of cutaneous lipids in a reconstructed skin model and human skin: spectroscopic imaging and chromatographic profiling”,
    Ali Tfayli, Franck Bonnier, Zeineb Farhane, Danielle Libong, Hugh J. Byrne, Arlette Baillet-Guffroy,
    Experimental Dermatology, 23, 441-443 (2014)

  • Jakub Wojcieszak added an answer:
    Why do MTT and XTT assays give inconsistent results?

    I have a problem in my cytotoxicity testing.

    I tested one compound on 3 various cell lines with MTT assay and it gives concentration-dependent decrease of mitochondrial function (by around 60% at the highest concentration) on all 3 cell lines.

    However, when confirming my results with XTT I see no effect, or even a slight increase of substrate reduction (by around 10%).

    LDH assay also points no increase in the number of dead cells.

    I repeated MTT with this compound in modifications of the method:

    1. Adding MTT into the medium -> incubation -> aspiration -> solubilization in DMSO -> shaking -> reading
    2. Aspirating the medium -> adding MTT -> dissolving in DMSO -> shaking -> reading

    ... and it always gives the same effect, so I guess it is not chemical interaction between my compound and MTT itself.

    Does anybody have an idea what can be the cause of this inconsistency? I know that MTT utilizes NADH to reduce, while XTT uses NADPH, so is it possible that my compound selectively lowers NADH concentration or inhibits enzyme responsible for MTT reduction?

    Jakub Wojcieszak

    I extended the incubation time to 72 hours and it resulted in stronger decrease in MTT and strong increase in XTT (153% of control group). Strange ...

    For now I am planning to measure intracellular total and reduced glutathione levels and mitochondrial membrane potential with JC-1.

    Do you have other suggestions or possible explanation of what I am experiencing?

  • Petr Mlejnek added an answer:
    Can % viability be considered an accurate representation for proliferation measure by MTT assay?

    My MTT assay shows proliferation in treated microglial cells as compared to noemal control. If one mesures using %viability, the values for the treated group come to 150% relative to the normal controls. Can the be considered as a valid representation? If not, what are the alternatives? should a direct representation of absorbance work fine?

    Also, could anyone suggest a better and more efficient proliferation assay, that is cost effective too.

    Thank you!

    Petr Mlejnek

    No. % viability means to discriminate live and dead cell usually based on permeability of cell membrane. This measurement fails to uncover early apoptotic cells which are in fact dead (but have intact membrane). This is really huge problem! On the other hand, MTT assay is not able to distinguish between the number of cells and the decreased viability. The decreased value of MTT reflects either the decreased number of cells or the cell death, or very often both events. However without further analysis you do not know what has happend.  


  • Khatereh Khorsandi added an answer:
    In my MTT test, when I use higher concentration of drug for treatment, there is higher absorbance than in the control. Can anyone help me?

    In my MTT test, when I use higher concentration of drug for treatment, there is higher absorbance than in the control. Can anyone help me?

    Khatereh Khorsandi

    thanks all, I made negative controls for all experiment but now I think the problem may cause from color of drug.

  • Hoda Abolhasani added an answer:
    Cells treated with DMSO show less viability than cells treated with the compound, how is that possible?

    I have performed a MTT viability assay to see how my cells (MCF-7 and HCT-116) respond to a drug. I had one control with cells + medium and another control with cells + medium + DMSO in different concentrations, the cells treated with the compound (wich is dissolved in DMSO) showed more viability than the cells treated with DMSO only in the same concentration. How is that possible? Is that normal to happen in a MTT assay?


  • Hussein A Abbas added an answer:
    Any advice on the UV to inflict DNA damage to HEK293 cells in culture?

    I am studying a protein and from imaging I can see that my protein is recruited to sites of DNA damage. I wish to UV irradiate HEK293 cells in culture prior to collection and analyses by RNA-seq and mass spectrometer. Does anyone have an idea of how to (protocol and instrumentation) UV irradiate cells in culture for such studies? 

    Hussein A Abbas

    I think an important issue to consider is whether you are interested in acute or chronic changes. If acute, you want to plan your experiments to maybe collect the cells within 6 hours. If you want to see changes over time, that's a different issue. We have done similar experiments in the lab but with different cell lines-

  • Coral Sh added an answer:
    Can DCFH-DA cause cell death when added to measure ROS level?

    I have been trying to measure ROS in mesothelioma cell lines using DCFH-DA. But every time after adding DCFH-DA to the cells even at 10 micromolar, the cells would become rounded and detach after 15min of incubation. I tried to reduce to 1 micromolar and then 0.5 micromolar. The cells were ok but the fluorescence is too low. Has anyone encountered the same problem?

    Coral Sh

    Thank you so much for all the comments and suggestions. 

  • Adeola Folasade Odewusi-Ehigie added an answer:
    Which is better: MTT or cell viability assay?
    I would like to determine the concentration of an anti-cancer drug on a cancer cell line to determine the concentration that I have to use to kill only half of the cells. I used a cell viability test before and checked with different concentrations of drugs. Then I choose the concentration that gave a viability around 60%.
    Adeola Folasade Odewusi-Ehigie

    I'll go for CCK-8, It's very easy to use, sensitive and straight to the point. With it you can determine the viability of your cells at varied concentration within a limited period of time. However, the Kit is expensive. In the alternative you can use MTT Assay which will take an extra hour before you acquire your results. All the best!

  • Gregory J Brewer added an answer:
    What cell viability assays for low concentration neuronal culture?

    I want to determine the level of toxicity of some treatments on neuronal cultures with a very low concentration (from adult animals - plated on Poly-Lysine coated coverslips - between 100,000-200,000 cells/ml max ).

    I already tried LDH assay kit but it seems that the density of cells is too low.
    I read that Alamar Blue or PrestoBlue are working with low concentration of cells. Anyone has any experience with that in neurones at low density?

    Also, as I am not able to plate my neurons in 96 well-plates (because they don't stick to plastic), I need to do the test in 12 well-plates containing coverslips with my neurones. And I am afraid the bigger volume is diluting the LDH (or maybe the Alamar blue as well). Do you have any opinion about that?

    Many thanks.

    Gregory J Brewer

    For years we have used a single cell fluorescence assay based on propidium iodide stain of dead cells and fluorescein diacetate for live.  See our 1993 paper or buy Invitrogen's Live-dead kit for 100x the price.

  • Falugi Carla added an answer:
    Is it possible for a cell to be viable while being PI positive?

    Propidium iodide (or PI) is an intercalating agent and a fluorescent molecule with a molecular mass of 668.4 Da that can be used to stain cells. When PI is bound to nucleic acids, the fluorescence excitation maximum is 535 nm and the emission maximum is 617 nm. Excitation energy can be supplied with a xenon or mercury-arc lamp or with the 488 line of an argon-ion laser. Propidium iodide is used as a DNA stain for both flow cytometry, to evaluate cell viability or DNA content in cell cycle analysis, and microscopy to visualise the nucleus and other DNA-containing organelles. It can be used to differentiate necrotic, apoptotic and normal cells.

    Falugi Carla

    If you need to introduce some molecules inside a living cell, you can try to use liposomes. When PI is inside the cytoplasm, it can stain also mRNAs.

  • Panduranga Rao added an answer:
    How do I monitor bacterial viability in vitro?

    I would like to investigate the intracellular viability of pathogenic bacteria within a host mammalian cell. Is there any relatively simple way to do this without lysing the host cell?

    Panduranga Rao

    CFSE is a good idea if you want to look for replication (cell division). In case of intracellular bacteria  probably it is of limited use as half of the divided bacteria may not come out. 

  • Puszko Anna added an answer:
    How to understand the concentration of a compound in articles when I read about proliferation and viability assays on cells?

    I am a chemist but I need to learn how to make some cell assays. I don't understand one thing; in articles I can found concentrations of tested compounds used for assays in e.g. 5 ug/mL or 5 uM. Does it mean that the concentration of the tested compound was like this in each well or is it a concentration of a solution of this compound used to make the assay, which means that the real concentration in the well is different because I added only a small portion of the prepared solution into the well with cells in the medium?
    This question might sound silly, but no one around me knows the answer...
    Thank you for any help.

    Puszko Anna

    Thank you all for fast reply and good explanation!!! Now I am sure that my thinking was correct. :)

  • Sven Buelow added an answer:
    Why microplate reader counter does not match with cell counting by bare eyes?


    I am doing a neuroprotective in vitro study on shsy5y cell culture. By using live/dead assay, it will stain live cells as blue, and dead cells as red. I used fluorescence microplate reader to detact the total fluorescence of blue and red. It gives me promising result in the experimental group compared to the control(more live than dead). However, when I count the cells by my bare eyes using fluorescence microscope, the live and dead cells in experimental group and control group look similar.

    I am wondering why microplate reader can detect such a contrast difference, but it seems indifference by bare eyes? 

    Does everyone who use microplate reader also experience?

    Sven Buelow


    Uwe may be right, and you must consider that the microplate reader reads both colors separately, whereas with your microscope, you will probably be using two excitation/emission filters and be looking at both colors at the same time, which means that the blue will be in all cells, whether live or dead, as it can permeate the cell membrane, and the red will be in dead or dying cells with damaged membranes. So, in effect, you will be seeing red superimposed on top of the blue, and this may not be as easy to differentiate if the cells are for the most part still dying and the red portion is thus weaker than the blue in the same cell.

  • Josefin illergård added an answer:
    How to handle fluorescence of nano fibrillated cellulose (NFC) scaffolds during Live/Dead staining?

    To evaluate cell viability in a 3D printed NFC scaffold, I want to use a live/dead assay to stain the cells (dyes: calcein and Ethidium homodimer). Unfortunately I get a background staining from the NFC scaffold (presumably due to DNA residues of the NFC). Therefore, I was wondering if anyone knows how to handle that issue or knows a different approach for a selective live/dead staining? (I do not want to break down the scaffold)

    Josefin illergård

    We are experience the same problem, but with cellulose fibres and bacteria.  Unfortunately I don't have any solution. We are instead using GFP modified bacteria, but of course you can't determine the viability then.

  • Mario Glynn Hollomon added an answer:
    Can someone tell me how to wash TC20 counting slides?

    I read in a previous post that a member washes and reuses his counting slides used for the Bio-Rad TC20 cell counter. Can someone provide a methodology used to wash the slides for reuse?

    Mario Glynn Hollomon

    Sumit and Okan,

    I like your responses. Many Thanks!

  • Mark Thompson added an answer:
    Is it possible for cells not to metabolize MTT labelling reagent?

    Hi all. I have performed an MTT cell viability assay and the vehicle controls do not seem to be metabolizing the MTT reagent. I am basing this on the fact that I definitely see live adherent cells in the control wells yet I am seeing a very weak purple colour - looking almost the same in colour as the wells where I have treated my cells with anti-cancer compounds. Is it possible that the cells are not metabolizing the MTT reagent? I am working with MCF7 cells.


    Mark Thompson

    Hi Alexis, it's true that different cell lines can metabolize the MTT at very different rates. In my experience it seems that some lines only give a faint purple colour even if you leave them for a longer incubation, whereas others look strongly purple in under an hour! I did notice the protocol you describe has a final assay volume of 210 uL/well, which would make it difficult to detect a faint colour when it's that dilute. It sounds like you might benefit from a lower final volume which will increase the intensity of your signal.

    There are a few ways of doing the MTT assay so here's what I suggest.

    You can add the labelling reagent and do the 4 h incubation the same as the kit protocol, then remove the media. Wash with PBS (carefully), remove that, then add 50 uL/well of acidified isopropanol (or DMSO), agitate the plate gently for a few minutes to dissolve all the crystals, then read on the plate reader. If you have a faint colour you can reduce the 50 uL of solvent to 30 or 35 uL to increase the concentration and get a better signal. This way should work OK but if you still have problems then you can try the most reliable way of doing it (in my experience).

    The other way of doing it is to remove the media and do a PBS wash (also remove this) before adding the MTT. Add 100 uL/well of the MTT at 0.5 mg/mL (you can make this up yourself, or if you dilute your premade labelling solution 1:10 in PBS this should be close enough). Incubate for as long as necessary for your cell line (up to 4 h but may be less; just keep track of it the first time). Remove the MTT solution, and blot the plate upside down on tissue to make sure all the solution is out of the wells (gently, to avoid the risk of detaching the cells). Then add the solvent, dissolve and read as above. I've found this final method is by far the most reliable and reproducible for the lines I've worked with but other people may have different experience; best advice is try it a few different ways and see what works best for your setup. Finally... Francis made a very good point so you may need to verify the compounds are killing the cells by other means. Good luck!

  • Ortrud Barth added an answer:
    How to check cell viability assay without killing the cells?

    I want to check the cell viability of 3T3 cells without killing them or deteriorating their proliferation. I tried CCK-8 kit which keeps the cell alive but it affects the cell growth and proliferation. Please suggest some assay where I can use it without killing cells. 

    Ortrud Barth

    Try it with cotton-blue

  • Nur Bashira Shaharuddin added an answer:
    How can I choose reliable seeding density before further to viability assay (MTT) for drug dosage dependence toxicity?

    Drug incubation more than 24 hours

    Seeding density that yields exponential or maintain viability after more than 24 hours of culture before drug incubation?

    Nur Bashira Shaharuddin

    Thank you so much for all replies! Really appreciate it

    Previously in my pilot studies, I used MTT assay on different seeding densities for required incubation hours ( I choose 72 hours based on papers and maximum exposure of experimental agent to the cells) . Most of lower seeding density ( not sure how to choose)  yield exponential pattern and started to decline after day 4. The highest seeding density however, yield constant pattern of viability (no decline) up to 4 days. I am not sure if I need to repeat my experiment using cell counting assay ( proliferation) instead  that would be more representative assay or stick to MTT assay. 

    I read studies that treat cells with mitomycin C ( to stop proliferation somehow ) in case it gives false positive result to inhibitory effect of drug ( cell died due to confluency instead of drug). I guess it also helpful to check confluence status as well like Jeff's said. 

  • Petra Pusic added an answer:
    Is it possible to observe P. aeruginosa for live dead cell counting by dm2500 leica microscope?

    I am afraid that sample might be easy to bleach by the light source

    Petra Pusic

    Should not be a problem. I have done it with LSM710 CLSM as I needed Z-stacks of my biofilms. DM2500 Leica is TLM if I am right?

  • Niraj Rajesh Bhatt added an answer:
    At what dose and time duration of treatment do AR42J cells respond to cholecystokinin in terms of cell death and UPR activation?

    I am treating AR42J cells with 10-8 M cholecystokinin (CCK) for different time intervals up to 8 hours. Following this, I need to check for markers of cell death and for activation of the Unfolded Protein Response (UPR). I have used Annexin-V staining, Alamar Blue Viability assay and Calcein-AM staining for cell death, and not found any significant differences between the treated and control cells.

    I did not find any significant changes in UPR markers as well. The markers I have checked are ATF6, GRP78, PERK, phospho-PERK, IRE1, phospho-IRE1, XBP1s, CHOP, ERO1L, OS-9 and PDI.

    I am right now checking the levels of pancreatic lipase and amylase in these cells after treatment with CCK which would be the most important marker to comment on acute pancreatitis like phenotype.

    I need suggestion for treatment that I should give to AR42J cells to hyper-stimulate zymogen production and secretion.

    Thanks a lot for taking time to read this and for your valuable suggestions.

    Best regards.

    Niraj Rajesh Bhatt

    Hi Terry,

    Thanks very much for your answer. Well, I missed out on informing that I have done a dose response from 10-6 to 10-8 M CCK, and time kinetics up to 8 hours. I have not seen significant or consistent change in number of dead cells in any of the condition. All higher time points are similar to 4 hour time point.

    Overall, I am not sure if these cells are suitable as a cellular model for studying acute pancreatitis, specifically the role of ER stress in acute pancreatitis.

    Thanks again for your response.

    Best regards.

  • Ayman Abuelela added an answer:
    When using resazurin to asses viability should I see the color start to change in viable cells immediately?

    I am trying to asses viability of Acanthamoeba using resazurin (Alamar Blue) .

    Ayman Abuelela

    It depends on the number of cells and cell type. 

    You may consider optimising the time taken to reach plateau using different number of cells before commencing the actual experiment.

  • Gaby R. added an answer:
    How do I reduce viability in resazurin assay for acanthamoeba?

    I have been trying to set up a viability assay for acanthamoeba with resazurin but very low absorbance has been seen. Im starting to notice some small crystals sometimes in the bottom of my T-75 cultures, I am worried this can be a sign of contamination, no other bacteria, fungi or microorganism can be seen in the microscope but im still not sure. Could this be the reason? The assay I have set up measures the absorbance at wavelenghts 570 and 600 reference.

    Gaby R.

    I added 10ul per well into a 96well plate with 100 ul of cell culture 

  • Vinay Sharma added an answer:
    cell viability interpretation issues in MTT Assay. Is compound cytotoxic or not?

    In MTT Assay, Cell viability is approx 50% when concentration of Toxicant is increased from 0 micro-molar to 50 micro-molar. But when the concentration is further increased from 50 micromolar to 500 micromolar the Viability is almost same, it hardly reached 40%. Can anybody suggest me, how to interpret the toxic behavior of compound used. Whether it will be considered cytotoxic or not?HeLa cells were used for MTT Assay.

    Vinay Sharma

    Dear Sunil,

    Thanks for the reply. Is there any specific software as you have mentioned "MTT Assay software". If it is so, can you please share the source?

  • Devan Thiran added an answer:
    Is it suitable to use Trypan blue dye and MTT Assay in determining percentage of cell viability instead of OD600 reading in alga culture?

    I'm doing treatment of heavy metal Mercury on marine alga (Chlorella vulgaris) and need to construct a dose response curve and also identifying IC50 value. But I'm now facing problem in getting the cell viability. Any ideas. Please do share with me. Tq.  

    Devan Thiran

    Tq for the answer Ameneh 

  • Rachel Elaine Hewitt added an answer:
    Is there anybody who has an established protocol for a 107a assay used for functional assays with human NK-cells?


    I try to establish a 107 a assay unfortunately without success. I used human PBMCs and cocultured them with several cancer cells (with and without developed drugs). The flow results looked like a full degranulation even when PBMCs were cultivated alone. PBMCs were isolated with standard procedures. Does anyone have an established 107a assay protocoll which could improve my results?

    Rachel Elaine Hewitt

    I incubate 2x105 cells in FACS tubes at 37oC, 5% CO2 with a CD107a mixture (10uL /ml CD107a (FITC conjugated & 0.7 uL/ml golgi stop (BD (554724) & relevant antigens or control antigens for 4 hours. After 4h wash & stain with surface antibodies & live/dead cell stain for 20 minutes, fix with 2% PFA. I find it works a treat for CTL & also degranulating NK, based on a paper by Mike Betts.

    u = micro

    Betts MR, Brenchley JM, Price DA, De Rosa SC, Douek DC, Roederer M, Koup RA. Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation. J Immunol Methods. 2003 Oct 1;281(1-2):65-78. 

    Good luck 

  • Robert James McClelland added an answer:
    Has anyone encountered interference of Fe3O4 with tetrazolium salts-based viability assays?

    Has anyone encountered any interference of Fe3O4 with tetrazolium salts-based viability assays? Thank you.

    Robert James McClelland


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  • Monika Toma added an answer:
    Which viability test is better ? MTT or trypan blue ?

    I am going to perform cell viability assay after treatment with inhibitors. I unfortunately have no possibility to use flow cytometry and have to perform MTT or trypan blue assay. Which one would you advice me to use?

    I have slight experience with both of them

    1. As I understand for trypan blue I have to count like 1000 - 2000 cells and count viable-dead cells proportion? But I don't know why I don't trust my eye (we don't have cell counter) as much as absorbance reader.

    2. On the other hand I don't have good experience with MTT cause of big differences in either wells that should have same absorbance value, as well as experiment repeats.

    Thanks for any advices. 

    Monika Toma

    I prepared cells for trypan blue test, and incubated cells with my inhibitors. My inhibitors have some influence at cell proliferation (they slow it). I cant use mtt, cause incubation with inhibitors results in different cell density. Data I'll receive wont be only viability but also cell density difference. Only way now is flow cytometry or trypan.

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