I  will recommend MTT assay  if you wanna detect cell viablity without adding drugs. But as you said big differences would occur when cell number is not equal. My experience is:  fist resuspend your cell, then dilute it to required concentration and pipptte same volume to each wells, finally, test your cell with MTT or CCK-8 following instruction. It should be noted that  bacterium polution will cause very big differences. So, take  good care of your cells and good luck.

Hi Monika,

Trypan blue cell exclusion depends on the integrity of the cellular membrane while MTT assay depends on intracellular activity to metabolize reagent and give a color for absorbance measurement. I suggest that Trypan blue would be used when checking your cell suspension's viability prior to plating. MTT assay would be suitable for your experiment after treating your cells with inhibitors. I agree with Liu in using CCK-8, as far as I know, you just have to add this reagent to your culture, unlike traditional MTT assay where you have to process cells first. You may also want to check this Promega's RealTime-Glo (see attached link). I hope this would help. All the best!

http://www.promega.com/resources/protocols/technical-manuals/101/realtimeglo-mt-cell-viability-assay-protocol/

i  agree Liu Bin... Cell Count  is very important and also MTT solution preparation you use PBS. it will give better results.

MTT assay is probably not a good test of cell viability. Even if you measure MTT reduction in a given viable cell population you will see great differences among cells, with heavily blue cells and cells which remain faintly coloured. I will prefer either trypan blue dye or measurement of medium lactate deshydrogenase release, an assay which is easily done in routine with microplate reader. 

Well I always did it like this. Firstly counted my cells, than diluted them to needed concentration and than I put them on 96 well plate. But as I said I had problems with catching the right moment for removing MTT and dissolving crystals. It was always ending with huge differences between wells that should have same value and I don't even mention differences between two separate experiments. 

Another thing is that substances I was treating cells with were brown-ish and by themselves added color after diluting MTT crystals. What is more! when I try to wash wells after MTT incubation with PBS, I think I was detaching some cells what again could result with different values. 

So firstly, I think that MTT is fine but not for chemicals like the ones tested by me (plant extracts). Secondly you have to take GREAT care bout same cell number in each well.

Those were the reasons that made me wonder if it wouldn't be easier to make it with trypan blue. When I think bout performing MTT now, I find it kind of suitable, cause of transparent chemicals. But still I'm so much worried bout cell number...

Hello Monika,

     I would suggest to go for MTT as you don't have cell counter otherwise trypan blue is better than MTT. In trypan blue exclusion method u have count several thousand cells and it is stress full and time consuming and more over every time manual error will be there and the manual error will be different each time. in case In MTT only contamination problem will give u a false reading in absorbent measurement. In MTT if contamination happened then that will gives u a added crystal so u will get a wrong absorbency. Otherwise If u want to perform some inhibitory effect to the cells MTT will gives you a better result than trypan assay.

But in MTT you have to make sure few things.

1. cell dilutions should be approx same for every plating times otherwise every times u will get a different absorbence for same concentrations.

2. MTT should be prepared freshly in PBS and better to avoid light.

3. take care that no contamination to the cell while plating.

4. Drugs/inhibitor dilution should be same or use same pipette for dilution each time other wise every time if u used different pipette u will get a variation cause every pipette have a own correction volume.

5. maintain the same timing for each time for cell plating or drug adding almost 24 hrs or after 48 hrs the adding MTT before 4 hrs each time u have to main same time difference otherwise u will get a variation in result.

6. after adding MTT to the cells do not keep for long time take the reading within time period it will give u a uniform reading.

7. use the DMSO high grade for MTT

Hope this will help u.

All the best.

I did all MTT, Cell titer blue (Promega), and Trypan blue cell counting with cell counter. They gave pretty same result but the Promega one saves your time. You obviously need the blank (medium and drug alone) to substrate the background. I would recommend you aliquot MTT 5%/PBS solution into 1-2 ml stored in the dark and -20°C. Best luck to you.

Siddik, reading what you wrote it seems I'm performing it perfectly fine. 

Trang, thats what I needed. To know that results from MTT and trypan are similar. I always use all controls, negative with MTT, negative without MTT, drug alone and positive with H2O2.

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.

Trypan blue is good test when few number of sample as tested.dilute cells if highly concentrated .

Freshly isolated small sized cell is tested by MTT .

I'll tell you what I figured out until now. I have long incubation (5 days) and MTT is not suitable for me because it shows changes not only in cell viability but also in cell density. Because my compounds inhibit cell divisions, I can't use MTT to evaluate cell viability. I have to use trepan blue + cell no. counter or propidium iodide + flow cytometry.

Are you using proliferating cultures and treatment time of more than ~4 h? Then please read this. Viability assessment with proliferating cultures requires single cell analysis and cannot be done in a 96-plate format using a plate reader. I wrote the following section for the most misleading crystal violet "cytotoxicity" assay which applies to all 96-well plate "viability" assays. A major advance in cancer research will be made when such misinforming and useless assays will be removed. "The Crystal Violet "Cytotoxicity" assay with proliferating cultures will determine anything but cytotoxicity. The 2016 protocol description by Feokistova et al. is for non-proliferating cells. Please see their Fig. 1 (control wells before and after treatment; there is no change in cell number!!!). In fact, the description of all such 96-well plate assays (MTT, CellTitre-Glo, etc) for viability assessment with proliferating cultures are majorly flawed. Misinformation perpetuated by misuse of such assays, as well as large-dye uptake assays (trypan blue, PI) have been discussed (PMID 30071623 and refs therein, including PMID: 27750219). If you use the single-cell MTT assay described in PMID 30071623, you will see that cells that are assumed to be dead (even with apoptotic morphology) are viable and metabolize MTT. Like the "gold standard" colony formation assay, all 96-well plate "viability" or "cytotoxicity" assays that are performed with proliferating cultures determine the ability of a test agent to convert proliferating (dangerous) cancer cells to dormant (most dangerous, tumor repopulating) cancer cells, and not to dead cancer cells. The IC50 values measured by such assays reflects a sum of cell cycle checkpoint activation (a pro-survival mechanism), short-term dormancy due to reversible apoptosis (anastasis), and early stages of durable proliferation arrest (dormancy, due to polyploidy, multinucleation and senescence, all of which contribute to disease relapse). None of these well-documented responses is associated with loss of viability. Viability assessment with proliferating cultures requires single cell assays and cannot be determined in a multiwell plate format using a plate reader."