Cell Viability Assay

Cell Viability Assay

  • Yilin Zhang added an answer:
    Will fluorescent assays (live/dead cell viability) be okay for my in vitro gold nanoparticle experiments?

    I want to see how effective my gold nanoparticles are at killing prostate cancer cells. Previously, I tried using an MTT assay to detect cell viability. However, there were a few problems with it because the MTT crystals formed from viable cells have a peak absorbance around 540-560 and my gold nanoparticles share a peak absorbance in this range as well. Not to mention, my gold nanoparticles are coated with epigallocatechin gallate (EGCg) which is an anti-oxidant that will likely react with the highly reductive MTT dye.

    Now I am looking into other methods, such as the live/dead assay which is a fluorescent assay. I am looking at this kit from life technologies and I want to know if this will work for my experiment. I plan on using a microplate reader, and the Ex/Em wavelengths for the live and dead dyes are 494/517 and 517/617 respectively. What are your opinions on this?

    Yilin Zhang · University of Chicago

    I think LDH and Trypan blue are feasible.

  • Dirk Schaefer added an answer:
    What are the difference between the MTT assay and the CFA assay? Don't the results tell you the same thing?

    What are the difference between the MTT assay and the CFA assay?

    Dirk Schaefer · TalkingCells

    Dear Jee Li,

    Answering your first question:
    there are differences between MTT assay and CFA !

    "MTT" is the acronyme for "3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid" (other tetrazolium salts might also be used);
    using the MTT assays, you measure/quantify the activity of enzymes, predominatly located in the endoplasmatic reticulum, reducing NADH to NADPH, i.e. the rate of glycolysis; in adition MTT is partially reduced by succinate-dehydrogenase, located in the mitochondria;
    => focusing all on one point:
    the MTT assay measures the cellular viability, as these enymes 'work best' if the cell is alive.
    This "viability testing" can be e.g. correlated to proliferative or cytotoxic effects of substance/stressors applied to the cell(s).
    for examplary literature for "CFA"-application see:

    "CFA" is (in general) the acronym for "colony forming assay", but is also used for "Complet Freud's Adjuvance"; an other acronym used instead of "CFA" is "CFU" ("colony forming unit");
    for this purpose variabel methods can be applied, some of fix grown cells e.g. by glutaraldehyde and staine them therafter using e.g. crystal violet;
    include fixat
    => in this concern focusing all on one point:
    the CFA is a general term for 'quantifying cell proliferation' looking on finally 'killed' cells.
    The number of colonies formed can be correlated to the proliferative or cytotoxic effect of substances/stressors applied to the cell(s).
    for examplary literature for "CFA"-application see:


    Answering your second question:
    the result(s) "quantification" of cells 'look like' the same, but may tell you different 'storries':
    depending on your primary question, why you want to 'quantify' cell numbers,
    the MTT 'tells abot' (analysis) the metabolic (i.e. enzymatic) activity of the cell (which in turn might also be ínfluenced by the substance/irradiation/physical/chemical 'stress' apllied to your cell;
    the CFA 'tells about' (analysis) the "grouping" off cells effected by the substance/stressors applied.


    => the selection of the (appropriate) assay depends on the question you consider to solve/answer.

    Good luck in selecting the most appropriate method



  • Gozde Akdeniz Skvortsov added an answer:
    How can I prevent all the cells from emitting fluorescence in the red channel in a Live/Dead Assay of Microtissue?
    I'm performing the viability Live/Dead assay on Microtissue (high cell density of cells - 250,000 cells/ mm3) using Calcein Am and Propidium Iodide and imaging them using a confocal microscope (488nm Exaltation Laser). I intend to finally count the number of live and dead cells using Imagej.

    Now, thought the experiment works perfectly well for cells on a glass slide, they won't work too well with the microtissue (Using a z- stack with 2um steps). The green channel cells show (99%) cells to be green. The red channel (Propidium Iodide) shows (100%) cells to be dead. Though you should just be able to 1% of the dead cells in the red channel. (Picture attached)

    How can I prevent all the cells from emitting fluorescence in the red channel? Am I using a high concentration of dyes? Or is it just hard to image cells in 3D culture?
    Is there any other assay for cell viability?
    Gozde Akdeniz Skvortsov · Sabanci University

    Hi Naveen, thanks. I  do not have any other option apart from the confocal so I have to give it a try with the confocal. Hope I will also find a way like you did :)

  • Engin Ulukaya added an answer:
    How can seeding an exact amount of cells in each well be achieved?

    I need to seed cells in a 96 well plate with very low variability in cell numbers from well to well, I used a multichannel pipette but still this is not very accurate, any suggestions to seed an exact number of cells in each well? And any recommendations for accurate robotic pipettes that are not very costly?

    Engin Ulukaya · Uludag University

    Follow Omer's suggestions and seed them in triplicate (for the mean value) and then nothing to worry about! All the world do the same. We are not concerned about the edge effect as long as you do not place the plate too close to the inner wall of the incubator (plus make sure it is well humidified).

  • Henry E Young added an answer:
    Which viability assays can I do to confirm chondrogenic differentiation from MSC pellets?

    Which viability assays can I do to confirm chondrogenic differentiation from MSC pellets? Cells were pelleted in micro centrifuge tubes and differentiated.

    Henry E Young · Mercer University

    35S incorporation will need to be addressed with respect to the amount of CS-PG, KS-PG, and CS/KS-PG in your chondrocytes. With the ability of the KS molecule to structure up to 5 sulfate molecules (it is variable dependent on age of the cell) it will be interesting to quantify. Alternatively, you might try 14C-glucosamine. It is more of a 1:1 with respect to the ECM components, and the half life is much longer, so you can use significantly less of the isotope for your assays.

  • James E Talmadge added an answer:
    Can anybody suggest the protocol for the interpretation of MLR ( mixed lymphocyte reaction) data using MTT assay to measure cell viability?

    I need to check for the MLR (Mixed lymphocyte reaction) for the particular cells using MTT assay. I have to co-culture the responder cells (CD8 T cells) from one mouse strain with the stimulator cells (irradiated APCs) from the another mouse strain. Since I am planning to measure it by MTT assay, what are the important controls which I should use? Also, after measuring absorbance at 570 nm, how should I interpret the results from that, i.e. how should I determine the % viability of each sample?

    James E Talmadge · University of Nebraska Medical Center

    MTT can measure cell proliferation but not viability.  Parameters critical to a MLR include, number of responding cells per well (150,000 works for us), ratio of stimulators to responders (1:3 to 1:10 is likely optimal) and time of thymidine addition, which varies with species.  I might note duration of co-culture with thymidine (I like overnight) is impoatant.  As pointed out MTT is supoptimal, although it can be made to work.  However, the stimulation index (SI) is generally low relative to the use of thymidine.  You don't have to isolate T-cells or CD4 or CD8s to make it work.  Note, you can use a one way MLR, where you irradiate the stimulators (most common) or a two way MLR (where both lymphocytes respond).  The one way MLR is most common and is easier to understand your results. 

  • Srinivas Chinde added an answer:
    What is the solubility of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) ?

    Does anybody know what is the highest concentration of a Stock solution of MTT (powder from Sigma ) in Culture Medium ? 

    I know from Sigma that they tested their powder up to 5mg/ mL and that in the handbook the solubility in water reaches 10mg/mL . But does anyone with high competences in chemistry or by experience know wheter I can dilute it also in Medium up to 10 mg/mL ?! 

    And do you filter it before use?! I would be very grateful for any precise explanation ! 

    Thanks ! 

    Srinivas Chinde · Indian Institute of Chemical Technology

    Dear Vanessa,

    Normally  In cytotoxicity assays we are using 5mg/ml of mtt as a stock. Dissolving solvent is PBS not to media ( becoz media interferes absorbency of final O.D). If u want to reduce the MTT dissolved PBS then you could add 5 ul (microlitre) per well sufficient.

    Before  added to the well you should cover the foil or to take amber color tube to preparing after that filter it with o.2 to 2 um filter it

    Finally make sure the seeded cells should be 5000 at the time of seeding the cells in the 96 well plate then will get the absorbency has to be in between 0.1 to 1 O.D.

    Absorbency depends upon the 1) Number of cells seeded to the plate

    2) type of test compound

    All the best and do the culture with contamination free

  • Adolfo Rivero-Müller added an answer:
    Can GFP be used to detect cell death?

    We want to evaluate a killing kinetic through GFP labeled bacteria in order to assess the bactericidal activity of plant compounds. Will GFP give us the detection of cellular death or chromophores will still be active after the bacterium has died?

    Is there a better methodology for this purpose?

    Adolfo Rivero-Müller · University of Turku

    I think you should read this article:


    There is some apoptosis sensor there. The detection might be a little cumbersome though.

    Good luck

  • Vanessa S added an answer:
    Is there a correlation between 1) cell size and metabolic activity specific 2) metabolic activity and MTT metabolisation ?


    1) I am performing MTT assays and was wondering how much the cell size can influence the mount of metabolized MTT?

    I thought that the amount of mitochondrial membrane, (so also the amount of active enzymes that metabolise MTT) correlates with cell size and therefore lets say 100 normal sized cells could metabolise approx. the same amount of MTT than 160 smaller cells, right?! 

    2) does anybody know if and how the metabolism of nutrients (Glucose, aa ) is connected to the activity of the MTT metabolizing enzymes ?

    As maybe the presence of nutrients at the time point of the addition of MTT !!! can influence the capacity of metabolizing the chemical .....  (I mean : 100 cells consumed a lot glucose --> remaining glucose in medium is less and metabolism goes down...so also the metabolism of MTT, ..... smaller signal,.... but treated cells, where only 50 are present consumed less Glu, therefore still more in the medium when adding MTT and therefore 50 cells can metabolize more than 50% of the MTT than 100 untreted cells) 

    Thanks a lot for reading and trying to understand and helping :) ! 

    Vanessa S · Campus IFOM-IEO

    Ok, thanks a lot !  

    I already thought it can only help me to get rough ideas about the survival ... although many labs are using it as THE method to prove the effect of a drug... 

    I also didn't know 

  • Jordan Robin Yaron added an answer:
    How do Hoechst 33342 and Ethidium homodimer coallign in live/dead cell viability assays?

    I'm running experiments using complement-mediated ablation to selectively destroy perisynaptic Schwann cells at the lizard neromuscular junction, and I was wondering if anyone had experience with using a Hoechst stain with ethidium homodimer to distinguish live vs. dead cells. When I look at the preps I've done so far under a fluorescence microscope, I see dead cells marked with the ethidium homodimer, but this fluorescence is not matching up with the Hoechst staining for those cells: they're either next to each other or in different planes, and do not take on the same shape or pattern. I do not think this is a result of the filters on the microscope being misaligned, but would like to know what would give rise to these different patterns and what I could do to improve my live/dead cell assay.

    Jordan Robin Yaron · Arizona State University

    Hoechst 33342 should be permeable to all mammalian cell types to my knowledge. Is it possible that your Hoechst has gone bad? Have you imaged your samples on a different microscope?

    Are you certain you have H33342 and not H33258? H33258 is much less permeable due to differences in hydrophobicity.

  • Amit Kumar Mishra asked a question:
    What is purpose of fixing the cells during IHC or NR assay?

    Fixation of cells is done by paraformaldehyde.

  • Kelly Mcgrath added an answer:
    Is normalization of MTT assay across multiple plates possible?

    Grad student with a "simple" problem.

    Im aiming to test the neuroprotective effects of a compound series against a neuroblastoma cell line, against a know neurotoxin. The general goal is to use a 24 well plate, and test cell viability via MTT. 

    Im running into a concern with confounding variables, and restricted space. is there an easy way to normalize my results across several plates? 


    cells + MTT

    cells + neurotoxin + MTT (negative control)

    cells + test compound + MTT

    neurotoxin + MTT

    test compound + MTT

    this leaves 2 trial runs for varying concentration of test compound, which is rather limited. 

    It would be easier to do my controls on a separate plate, and my experiment w/ a cells+MTT control on a separate plate, and then normalize the data.

    Any suggestions? 24 well plate was picked because of differentiation stipulations that need to be addressed before exposures

    Kelly Mcgrath · University of Montana

    Fantastic. Thanks for the advice Dr. Pool!!

  • Hardik Patel added an answer:
    Can I fix cells in formaldehyde or after prodidium iodide staining to measure cell viability by Fluorescence microscopy or FACS?

    I want to measure viability of bacterial cells after treatment with antibiotics. So I stain bacteria cells with Propidium iodide after antibiotic treatment. However, my flow cytometry lab will not accept my samples because they are potentially hazardous CAT 2 bacteria. Fixing the cells in formaldehyde after PI treatment is not an option as this will interfere with PI staining. Please any ideas or alternatives? Thanks.

    Hardik Patel · Nirma University

    Hi Linda,

    The following paper might help you for your problem.

    Caruso, G., M. Mancuso, and E. Crisafi. 2003. Combined fluorescent antibody assay and viability staining for the assessment of the physiological states of Escherichia coli in seawaters. Journal of applied microbiology 95: 225-233.

  • Joaquín Jordán added an answer:
    Which is the best method to measure ROS and cell viability in same plate?

    Thank you. 

    Joaquín Jordán · University of Castilla-La Mancha

    Dichloroethidine alone should work. Increases means ROS production and then it binds to DNA, and using PI channels with a FACS machines you can follow the DNA state.

  • Carl Manner added an answer:
    Is APH assay reliable and better than MTT in determining cell viability?

    I am performing experiments on 3T3, caco2 and HeLa cell lines. Does APH assay have any advantage compared to MTT assay in cell viability studies?

    Carl Manner · University of North Carolina at Greensboro

    I've used MTT for proliferation studies in those cell lines, and it works well. For viability I always used trypan blue and flow cytometry - it's nearly foolproof (and inexpensive)

  • Durgesh Pitale added an answer:
    How do I conduct an NBT test of neutrophils ?

    to know the phagocytosis of neutrophils

    Durgesh Pitale · National Institute of Immunology

    Hey Sarjan,

    What type of assay do you want to perform i.e. colorimetric or microscopy or FACS? Are you using purified neutrophils or complete blood smear?

    Refer papers which suggest protocol for NBT test in Chronic Granulomatous Disease.

  • Can IC50 determination by MTT be used for showing cell viability?

    MTT assay was performed on various cell lines and IC50 was calculated. However, cell viability was not performed separately. Is it logical to show by any means that compounds under study were not affecting cell viability if IC50 values are larger (less potent)?

    Paulo Michel Pinheiro Ferreira · Universidade Federal do Piauí

    If you only just want to determine IC50 values, MTT is a good tool. But if you want to study mechanisms of action,  MTT does not work since it is an indirect way to quantify viability

  • Ru-Jeng Teng added an answer:
    In cell culture, what is the appropriate solvent for a drug other than DMSo?

    In cell culture, while performing antioxidant and cell viability assays what is the appropriate solvent for dissolving a drug. In general, DMSO is used as a solvent for dissolving but what solvent other than this can be used as a solvent and at what concentration it should be used? It would be greatly helpful if anyone tells this with reference.

    Ru-Jeng Teng · Medical College of Wisconsin

    When I was a fellow I was told ethanol has some weak antioxidant activity but I did not check into detail myself. I might be wrong but just learn not to use it when I did the free radical experiments.

  • Sylvestar Darvin added an answer:
    Is anyone familiar with performing MTT assay for Plant extracts?

    MTT assay concentration fixing stock solution perpetration and require volume for test.

    Sylvestar Darvin · Loyola College

    thank you very much

  • Nandaki Nag added an answer:
    How can we discuss about the results when both cell viability and mortality decreased?

    Dear friends

    I have cultured hella cells and the cell mortality and viability have been checked under treatment with special drug. So, I have seen that the cell mortality and viability decreased together and I want to have a conclusion about the effect of my drug on hella cells. can you help me?

  • Nagendra sastry Yarla added an answer:
    Is the MTT assay suitable for antibacterial activity?

    Thanks in advance for your replies.


    Use this article for reference.

  • Manikanta Murahari added an answer:
    Can anyone suggest a protocol for MTT or SRB assay of nanoformulation?

    We have prepared a nanoformulation of anticancer drug and performed MTT and SRB assay of API, nanoformulation and placebo (nanoformulation without drug) at concentrations of 5-100 ug/ml. 

    OD readings were observed at a range of 2-3 for all the concentrations for MTT assay. We thought that MTT is interfering with the formulation components and tried with SRB assay.

    In SRB assay, we found OD of placebo was more than control

    For nanoformulation OD readings were also more than control but less than placebo

    Can anyone suggest the interpretation of results or share any protocol for the same



    Manikanta Murahari · SVKM’s Dr. Bhanuben Nanavati College of Pharmacy

    We also tried to wash with PBS, because of incubation for 24-48 hrs the particles are settling on the plate and we couldnt wash the wells efficiently. 

  • Dr Stephen A. Butler added an answer:
    Why does my MTT is not changing color to purple?

    I am trying to determine the IC50 of an alcoholic mushroom extract in MDA-MB-468 cell line.  I can see the purple crystals under the microscope after the incubation with MTT, But after putting the solubilize reagent the solution doesn't change to purple color, it remains yellow and when I read the absorvance the values are very low near to the blank.

    I have the MTT kit from the ATCC and I use the following protocol:

    1. Seed 4,500 cells in 96 well plate. Incubate for 24 h.

    2. Replace medium with medium with my different concentrations of the extract. Incubate for 24, 48 and 72 h.

    3. Remove medium with the extract and replace with 100ul of fresh medium.

    4. Add 10ul of MTT reagent and incubate for 2:30h.


    Dr Stephen A. Butler · MAP Diagnostics Ltd

    Switch detergent, your cells are not lysing. We used DMSO with stubborn cells. As long as you treat your controls the same it shouldn't matter what you use to lyse.

  • Warren Viricel added an answer:
    Does phospholipid interefere with Alamar Blue assay which gives you false positive results?

    I have been working with liposomes loaded with cytotoxic drugs. I used homemade resazurin solution (Alamar Blue) to investigate cell cytotoxicity of my drug-loaded liposomes. I consistently obtained false positive results. For example, from Alamar Blue fluorescence reading, liposome-treated cell samples have 200% cell viability when normalized to untreated cell controls.

  • Pavel Montes de Oca B added an answer:
    Is it true that the MTT assay accuracy applies only down to 30-10%?

    I found a PPT online (citation missing unfortunately), where a comment mentioned that MTT assays are only reliable until a surivival of log1-1.5 which would correspond to 10-30%. The context was: Determination of survival of cell lines after irradiation by x-rays. Apparently MTT assays are fit to distinguish the radiosensitivity of cells but are only so accurate in determining cell survival. Does anybody know if this is true? If so, what is the reason for this and is there literature describing this phenomenon?

    Thanks for your infos

    Pavel Montes de Oca B · Instituto Nacional de Neurología y Neurocirugía

    MTT does indeed measure mitochondrial functionality no necessarily viability as it was originally described, although there is debate about this because even some other enzymes may reduce MTT not only mitochondrial ones. The density effect may be related to mitochondrial function that may depend upon cell contacts, this is only a guess. 

  • Pedro Cunha added an answer:
    What is the most correct formula to assess cell viability with Alamar Blue assay?

    We add resazurine to the following conditions:

    Just media (neg Control)

    Non-transfected cells (our positive control)

    Non-transfected cells + drug

    Transfected cells

    Transfected cells + drug

    To calculate the cell viability using Alamar blue we are measuring spectrophotometry at 570nm and 600nm

    We are using the following formula to obtain % cell viability relative to positive control cells:

    100x ((ΔAbs sample)-(ΔAbs neg CT)) / ((ΔAbs pos CT)-(ΔAbs neg CT)),

    and ΔAbs = Abs(570nm) - Abs(600nm)

    My rational is that:

    If (Pos CT-Neg CT) -------- 100% viability

    Than (Condition-Neg CT)----- x

    Is that to simplistic?

    Because I have read this info:


    and I indeed can find a formula that looks alike this one but only for fluorescence.

    Should we read fluorescence instead to calculate the % of viability relative to control cells?

    Do we really need to introduce the values of the Extinction coefficient of the reduced/oxidized form of resazurine in both wavelenghts?

    Thanks in advance

    Pedro Cunha · University of Coimbra

    Thank you everyone for your aswers, they were really helpful!

    We indeed always add the resazurine to a new culture medium. 

    For now on we will start to use fluorescence instead and use a formula that divides the RFU (condition) by the RFU(positive control, cell with no drug, non-transfected)

    Best wishes,


  • Mubin Tarannum added an answer:
    Anyone familiar with LDH assay and MTT assay?


    I run both cell viability using MTT and membrane integrity using LDH analysis, but I find the same compound shows higher levels of LDH release compared to the cell viability data. Can anyone explain this for me please?

    Mubin Tarannum · North Carolina Agricultural and Technical State University


  • Pradyumna Mishra added an answer:
    How can I isolate and culture lymphocytes for MTT assay?

    I have tried isolating and culturing lymphocytes by using density gradient centrifugation and treating the cell with nanoemulsion after letting it grow for 24 hours but after adding MTT I am not getting positive results as I can see only clumps of purple color not the entire well is purple even in controls.

    Pradyumna Mishra · Dr. Harisingh Gour University

    I agree with Patrick.

    Alternatively, if you have a cyto-centrifuge at your lab, you can prepare a mono-layer and visualize the cells under a phase-contrast microscope. Perform a qualitative assessment and score the number of positive cells. Although this might not address your issue comprehensively, but will help you to get a differential insight of control and treated cells.

  • Danilo Malara added an answer:
    How do I calculate the integrated formula for a bacterial viability stain?

    Hi all,

    I'm using the live/dead stain and would like to have information on how to plot the fluorescent signal that I got.

    In the manual (fluorescence micro plate reader section) they say to do the integrated intensity of the emission wavelengths of green signal (530+/- 12.5 nm) and red signal (620+/- 20 nm). Then they say that the excitation wavelength should be at 485+/- 10 nm.

    I guess that I have to calculate all the wavelength in the emission spectra from 515nm to 650 nm with 1 nm step (just to have more data). This spectra should be calculated for each excitation wavelength (from 475 to 495 nm).

    I probably found the formula for the integrated of the emission wavelengths, but I still not getting how to do the one for the excitation wavelength.

    My question is: how can I calculate the integrated of all this data? Does anyone that has used this staining kit have the answer?


    Danilo Malara · James Cook University

    Thanks Gerco for your reply,

    the problem is they say 530 nm +/- 12.5, but they do not say how accurate the reading shoul be. In other words, i can assume that they calculate accuracy of 0.5 nm or 1 or 10 nm.....this mean that i can have more or less fluorescent signal to integrate. So if i use 1 nm accuracy, i will have about 25 wavelength to integrate, while if i use a 12.5 nm accuracy i will have only 2 and result very different.

    I think i will send an email to the manufacture company and see if they can help on this.


  • Inaki Azcona added an answer:
    What techniques are available to study the viability of arbuscular mycorrhizal spores?

    I am using Tetrazolium (INT) as a viability stain for AMF spores but I am finding some inconsistencies in the results I obtain. Any other technique that I could use to contrast my results with INT? Thanks for your answers.

    Inaki Azcona · Koppert B.V.

    I am already conducting bioassays to study the inoculum viability. However, I was thinking in some other way to analyze it, less time-consuming. I think I will try to develop an alternative technique... ;) Thanks a lot for all your answers.

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