Absorption spectrum of formazan depends on pH and cell density at the endpoint. At pH 7, there are two maxima: 500 nm and 570 nm; the maximum at 570 nm is rather shallow and not good for the assay, while maximum at 500 nm is somewhat better.  On the other hand, at pH 10.5 there is a single maximum and much higher absorption coefficient at 570 nm. For this reason, it is advised to adjust pH of the solution with 0.1 M glycine buffer (pH 10.5). It seems to me from your description that you are measuring absorbance at lower than optimal pH. If you remain at pH 7 and wavelength ~500 nm, you will have much lower assay sensitivity, i.e. much lower ability to detect differences, and a lower analytical signal, which will force you to use higher initial cell densities. The higher initial cell densities reduce time window during which cells in the wells can grow exponentially, which is often desirable for these experiments. 

Plumb JA et al. Effects of the pH dependence of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide-formazan absorption on chemosensitivity determined by a novel tetrazolium-based assay. Cancer Res. 1989;49:4435-40. 

Absorption spectrum of formazan depends on pH and cell density at the endpoint. At pH 7, there are two maxima: 500 nm and 570 nm; the maximum at 570 nm is rather shallow and not good for the assay, while maximum at 500 nm is somewhat better.  On the other hand, at pH 10.5 there is a single maximum and much higher absorption coefficient at 570 nm. For this reason, it is advised to adjust pH of the solution with 0.1 M glycine buffer (pH 10.5). It seems to me from your description that you are measuring absorbance at lower than optimal pH. If you remain at pH 7 and wavelength ~500 nm, you will have much lower assay sensitivity, i.e. much lower ability to detect differences, and a lower analytical signal, which will force you to use higher initial cell densities. The higher initial cell densities reduce time window during which cells in the wells can grow exponentially, which is often desirable for these experiments. 

Plumb JA et al. Effects of the pH dependence of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide-formazan absorption on chemosensitivity determined by a novel tetrazolium-based assay. Cancer Res. 1989;49:4435-40. 

Dear Roman,

I read the article. As mentioned in the article, high number of cells have maximum absorbtion at 510 nm and I had high cell numbers too (from 100,000 to 700,000 cell/well) and I think that is why I had higher absorption at 492nm. An alternative is adding glycin buffer (pH 10.5) and reading at 570nm which I will try it probably.

Any idea regarding referencewavelength?

Dear Mariam: single wells in 96-well plates normally yield 30,000-40,000 cells  per well of confluent culture, so if you are using 96-well plates, your cell numbers are high and your culture is superconfluent. This may not be appropriate for most applications of MTT assay and you may be far beyond assay linearity. Concerning reference wavelength: 650 nm and higher are safe. Absorbance of formazan is rather low at 650 nm and you can verify it yourself: try 650 nm, 690 nm and compare (the difference will be small). 

Dear Roman,

Thank you for your sincere help.

I add two articles which use 100,000 and 200,000 of PBMCs / well.

I agree with you that 400,000 and higher amount of cells/ well in a 96-well plate give a super-confluent culture, but I read that appropriate OD in MTT assay is about 0.75 - 1.25 and my conditions which contain 100,000 and 200,000 cell/well had low ODs (about 0.25- 0.35). Because of that, I use high cell numbers.

Should ODs  really be about 0.75 - 1.25 in MTT assay, or lower ODs are accepted also? I see that some researchers in researchgate said that ODs between 0.2 - 0.7 can be good too. 

https://www.researchgate.net/post/Why_in_MTT_assay_do_we_measure_the_optical_density_OD_at_two_different_wavelengths1/1

You have not indicated before that you are using PBMC. These are not adherent cells and my advice about cell numbers is relevant to growing adherent cultures reaching confluence. Anyway, I suggest not to follow articles too literally, and try to optimize your MTT assay with your cells: load different amounts of cells, incubate, develop formazan, and plot absorbance vs cell number curve. Optimization of assay conditions (pH, wavelength) should be also done, unless you are using a validated assay for specified MTT solvent, dissolving reagent for formazan and buffers (pH). To avoid this optimization, you may want to buy optimized commercial MTT kit, but you still need to optimize cell numbers.

MTT assay looks simple, but there are numerous nuances, which are often ignored, and which can considerably affect the assay outcome. There is just too many variables: do you centrifuge plates and remove supernatant before dissolving formazant?  Do you use DMSO or surfactants to dissolve formazan? There is also question of biology, not just assay technicalities: is the experimental effect, which you want to see, affected by large cell densities? Do your cells tolerate high cell densities during the time window of  your experiment? You need to find answers to these questions. When you get commercial MTT assay, you can ask technical assistance to guide you through certain steps. 

The question about maximum absorbance is again related to the assay optimization. You need to perform the optimization of cell numbers and see the highest absorbance at which your assay is still linear. Whether this is your target absorbance depends on what you are actually doing; for example, if you are stimulating cell proliferation, you need to use conditions under which your untreated cells show lower absorbance than this maximum absorbance. Please note that if your maximum absorbance for assay linearity is, for example,  1.0, then values below 1.0 are safe, just try to keep wells with maximum number of cells at ~1.0. 

Dear Roman,

Many thanks for your advice.

Good afternoon. I would ask for your opinion with the following: I plant 5000 cells per well, I let them adhere for 24 hours and I put treatments for 72 hours. Then I verify in a microscope the results and I observe that in the high treatments there are very few cells, close to 10%, and in the controls the wells are at 90% but when performing the MTT technique, the wells that apparently did not have almost no cells give me about 50% absorbances compared to the control. I appreciate your comments.

570 nm

Yes, I reading at 570 nm. Also proved 450, 540, and 600 nm and the answer is similar.

Dear Miguel,

Two questions:

1) Do you remove each well supernatant before adding the MTT dye solution or not?

2)What is your treatment? Dose it have any solvent or not? If yes, you should include a control in your experiments which treat your cells by that solvent alone. Maybe it reduces the dye and confounds the results.

570-630 nm wavelengths.

Dear Maryam Zare.

First response

Yes, I eliminated the supernatant from the well before adding the MTT.

Second response

My treatment has DMSO as a vehicle, but I am putting the wells with DMSO as a control.

By the way, I was solve it by changing SDS by DMSO to dissolve the crystals.

yes I think using DMSO as a vehicle control is appropriate as well.

540 nm works well

570 nm and 650 nm.

https://www.atcc.org/~/media/DA5285A1F52C414E864C966FD78C9A79.ashx

Dear Miguel,

you could add a control to know whether the remnant drug interacts with MTT. It would be a control with culture media + drug, in the absence of cells.