The MTT assay may sometimes be a headache depending on the compound(s) used (e.g. vitamin C or NAC) due to their possible reducing activity on the MTT compound, which leads to a false positive viability (although the cells are dead or almost dead, or even no cell at all!). Or if the cells undergo apoptosis, then they will still be able to reduce the MTT compound (to produce formazan crystals) to some extent, thereby the assay may still consider them as alive cells but they would actually be dying. To overcome this problem, we use an ATP-based assay and its results are much more correlated with the cell images (proliferation or death) observed under the microscope unless your compound has no interference with ATP production (it is rare). ATP assay is also able to detect much lower number of cells (even down to 20 or less!) due to the luminescence' great sensitivity.

Positive control for cytotoxicity is the cells treated with a cytotoxic drug/chemical + MTT + solubilizing buffer (10% SDS in 0,1 N HCL in our case)

Negative control for cytotoxicty is the cells left untreated + MTT + solubilizing buffer.

Blank is medium with NO cell + MTT + Solubilizing buffer

However, this terminology if flexible. People mainly use two terms in viability assays; control and blank. If you say CONTROL, this means the untreated cells (+ MTT + SB). If you say BLANK, this means medium with no cell + MTT + SB (as above).

Importantly, the results of the MTT or ATP assay should NOT be given/interpreted as CYTOTOXICITY (as some people wrongly do!) unless you observe the cell death under microscope because they ONLY give you an idea of viability of the cell population. The viability might ALSO have been reduced by a cytostatic effect, or antiproliferative effect. These two effects should NOT be defined as cytotoxicity but some people unfortunately put them under the category of cytotoxicity. The term of viability should be a general definition comprising these three parameters (cytotoxic effect, cytostatic effect, antiproliferative effect). So, any decrease in viability may be resulted from either of these three effects!... All the best.

Protocols differ on the need for blank, negative control, or 100% death control. My recommendation is 100% death control of 100% dead cells+MTT reagent+Vehicle (if DMSO is vehicle for you). Dead cells can be accomplished by 0.1 to 1% Triton X-100, or any toxic compound which does not affect MTT reduction.

Here's a simple way to calculate % cell viability:

1) Calculate average of all 100% death control wells

2) Subtract result from #1 from all positive control and experiment sample wells (all control and treatment groups) - now background, non-live cell dependent color conversion has been eliminated

3) Calculate average of all positive control wells (100% live cells)

4) Divide all positive control and experiment sample wells (all control and treatment groups) by result from #3

5) Now all positive control wells will be close to 1.0 or 100% cell viability, and all treatment groups are relative to that. 0.5 = 50% cell viability.

6) Note that MTT is metabolic and enzymatic reductive capacity, which correlates with live/dead cells, but there are compounds which can suppress MTT without killing cells. Look at your wells under a scope and note the relative levels of dead or floating cells to confirm, before starting your assay.

I´m wondering about so much scientists prefer MTT-Assay, because neutral-red Assay are much more practicable.

I agree with Jochem: neutral red is an old test, but should be reappreciated. Moreover, it gives you the opportunity to observe your cells before lyisis and to catch an image of stained lysosomes.

Moreover neutral red isn´t genotoxic , good soluble (water), you see effects after few minutes (normal staining time 2-3h), rare inderaction with substances. I use neutral red for differend kind of cells e.g. 3T3- fibroblasts, MoDC .It works very well.

I agree with Jochem s well, but not so many news guys used it, and I do not know why. My students just do MTT, MTS and CCK8 assay although I love to have neutral red too.

I can really recommend to use different assays. My whole last year I compared neutralred and MTT (actually it was XTT) on my colon cancer cells. Neutralred led to results I favoured but the same experiment done with XTT turned my whole world upside down.

After that I switched to sulforhodamine B assay which is kind of a quantification method for total protein in the cell (roughly described). I had the chance to use another method, a rather expensive one (don't want to advertise it now but) based on the quantification of ATP of metabolically active cells (using luminescence) - and the results were quite reproducable.

After neutral red staining you can add at all times a protein staining for example kenazid blue

Totally agree on that. Or stain nuclei using Höchst.

Thanks y'all, quite useful. Especially Gin's response. Thanks

Positive control = active compound(eg :doxorubisin,etoposide etc..)+cells+MTT

Blank=cells without compound+mtt

Negative control= without drug medium+mttt

Calculation:

Cell density= OD sample-OD blank (MTT plus acidic isopropanol). Subtract the blank OD from each sample OD, then average the triplicate sample ODs. Set the sample OD from your untreated cells to 100%, then calculate the percentage of cell density for treated cells (x) by following ratio: OD untreated sample/OD sample = 100%/x.

Dear, Blank means background. I mean media+MTT+DMSO.

This way you can ignore effect of media absorbence. However you can use DAPI staining. its protocol is so easy and just take 5 min.

This may be a little off-topic, but in general MTT assays are not the gold standard for cytotoxicity as they measure metabolic activity. Thus, cells that are destined to die, but are still metabolically active register as live. Conversely, because some treatments induce growth delay, MTT and other colorimetric assays can detect this and be interpreted incorrectly as cytotoxicity, when it is simply a reduction in cell number due to inhibited proliferation. The gold standard for cytotoxicity is the clonogenic assay.

Agree with E.B. Different authors refer to MTT-type assays as measuring either viability, growth or proliferation; whereas what it really measures is (roughly) total metabolic activity of a culture. This (depending how long you treated the cells) is a tangled mixture of proliferation rate, death rate, cell size and metabolic activity per unit cytoplasm, any or all of which a treatment might alter. Suggest BrdU for proliferation rate, cell counts vs total protein for cell size, and dye exclusion or (yes), colony formation for % viable cells. Although some cell types will not grow from low plating densities even when viable. And other cells (eg neurons) may be alive but not proliferate at all. So you need to think what you really want to measure.

The MTT assay may sometimes be a headache depending on the compound(s) used (e.g. vitamin C or NAC) due to their possible reducing activity on the MTT compound, which leads to a false positive viability (although the cells are dead or almost dead, or even no cell at all!). Or if the cells undergo apoptosis, then they will still be able to reduce the MTT compound (to produce formazan crystals) to some extent, thereby the assay may still consider them as alive cells but they would actually be dying. To overcome this problem, we use an ATP-based assay and its results are much more correlated with the cell images (proliferation or death) observed under the microscope unless your compound has no interference with ATP production (it is rare). ATP assay is also able to detect much lower number of cells (even down to 20 or less!) due to the luminescence' great sensitivity.

Positive control for cytotoxicity is the cells treated with a cytotoxic drug/chemical + MTT + solubilizing buffer (10% SDS in 0,1 N HCL in our case)

Negative control for cytotoxicty is the cells left untreated + MTT + solubilizing buffer.

Blank is medium with NO cell + MTT + Solubilizing buffer

However, this terminology if flexible. People mainly use two terms in viability assays; control and blank. If you say CONTROL, this means the untreated cells (+ MTT + SB). If you say BLANK, this means medium with no cell + MTT + SB (as above).

Importantly, the results of the MTT or ATP assay should NOT be given/interpreted as CYTOTOXICITY (as some people wrongly do!) unless you observe the cell death under microscope because they ONLY give you an idea of viability of the cell population. The viability might ALSO have been reduced by a cytostatic effect, or antiproliferative effect. These two effects should NOT be defined as cytotoxicity but some people unfortunately put them under the category of cytotoxicity. The term of viability should be a general definition comprising these three parameters (cytotoxic effect, cytostatic effect, antiproliferative effect). So, any decrease in viability may be resulted from either of these three effects!... All the best.

Thanks a million Engin. Very helpful! I'd like to know how to interpret absorbances of treated cells that are lower than the blank (medium, mtt, dmso), resulting in net negative absorbances.

Hi Ubanako, In that case, the MTT assay should NOT be considered as the proper choice. There seems to be some interfering activities in your experimental system. So, you had better to go for the other assays.

Hi Ubanako: I have enjoyed reading the discussion following your question and agree with the comments regarding the use of metabolic assays, the cells being tested and the various controls that can be run. We use the resazurin and MTT assays in our work, both are metabolic assays and neither are perfect. I actually have a preference for the resazurin assay because it not cytotoxic so the same culture can be read multiple times which reduces costs. We usually follow the cells daily for 5-7 days after experimental treatments to obtain an initial impact on relative viability, cell loss due to apoptosis and proliferation. Clonogenic, trypan blue exclusion or specific function assays can be used to confirm viability results. Apoptosis assays should be used if you see a dip in assay results on days 1-3 or to balance cell loss against proliferation, and assays of cell number (actual counts, DNA or RNA quantitation) for confirmation of proliferation.

Hy, Bhuvanesh,

In what field you want the formula and the calculation ?

Can anyone provide me the good protocol for MTT assay with drug dilution and calculation.

 MTT assays are not a gold standard measure of cytotoxicity.  The endpoint is a surrogate endpoint and not necessarily measuring cytotoxicity (eg. growth delay and metabolic alterations give a signal), and as mentioned above, the assay is subject to artifacts due to treatments. Linearity of the response needs to be established for each cell line prior to using this method, and even then it is inferior to clonogenic measurement. As an editor, I reject manuscripts that use this measure alone and claim cytotoxicity as an endpoint.

 Thanks Dr.Eric J Bernhard.This comment is very valuable

Hello,

Do I have to add DMSO to the control wells (untreated cells), and if I have to, what is the amount that I should add?

However, the amount of DMSO that am adding with the highest concentration is 0.1microliter.

Best never to add undiluted DMSO to a cell culture as it will immediately kill cells locally that it touches, before mixing in, leaving a "bald patch". So mix the DMSO with part of the medium (such as 50% or 10%) before adding.

If you are having only one solvent control then yes, you would choose the concentration of DMSO (or whatever solvent) that matches the highest concentration that you are adding with your test substance(s).

thank you Dorothy

I agree with Jochem: neutral red is an old test, but should be reappreciated. Moreover, it gives you the opportunity to observe your cells before lyisis and to catch an image of stained lysosomes.