Hi Terezie,

I've some questions. What proliferation assay have you used? The number of cells seeded in the wells was the same? Time of incubation with the reagent was greater than 2 h? What amount of reagent per well? Have you try measuring the absorbance at 490 nm?

Hi María,

in my case I would not say it is proliferation assay, but "survival assay", since I test if my insulin compounds save the cells (primary rat astrocytes) from dying from hydrogen peroxide stress. The number of cells is exactly the same for the whole plate. I incubate the cells after overnight treatment with MTT for 3 hours. I have not tried to measure the absorbance at 490 nm, but from the picture you can see that some of the wells are completely blue and it was not because there would be more cells or they would live better. They looked dead to me as the other yellow wells, but there was a lot of formazan formed.

I am basically looking for an answer what can cause MTT to be transformed to formazan, if it is not the cells doing it.

Ok, yes, a survival assay. So, the problem is some wells that apparently seems having dead cells are inducing more formazan and not in accordance with the apparent number of cells you see. But, in these specifically wells, cells could have had any different treatment in some aspect, that could have induced a greater enzymatic activity, in spite of the apparent aspect of dead? You could repeat the assay in the same conditions but, before adding the reagent, count the number of viable cells by trypan blue, to find out if actually there are differences in the number of cells.

María Luisa Caballero thank you for your input! I am not sure I could do that because I cannot move the cells from the well and during the whole assay there is alway liquid in the well (I have found out thatthe formazan crystals are also in the media, so I cannot remove it). Also I feel it would interfere with the measuring, because trypan blue has very similar colour to formazan.

Hi Terezie,

What I wanted suggest you was the possibility to repeat or perform a test under exactly the same conditions as in the first assay (number of cells seeded, treatments and the same experimental condition for each well) but in this case, only repeating the conditions corresponding to two wells of those that have turned out yellow and two of those that have turned out purple, and before!! of adding the MTT reagent, counting* the cells and note the number of actual viable cells in each situation previous to MTT and subsequent formation of formazan. Then, follow the protocol by adding the reagent and developing the MTT assay. In this way, you would check if there is really a huge difference in the number of viable cells between the two situations, that could explain the differences in the formazan produced.

* To count the cells using trypan blue there are two options depending of the cell culture used, suspension or adherent. If you are using a suspension culture, I would mix gently and take 10 microL of the suspension and pass into an eppendorf with 10 microL of trypan blue, and count the viable cells. If it were an adherent line, I would first pass the supernatant medium from the well (in which the dead cells would go) to an eppendorf, and then I would detach the cells adhered to the surface of the well using trypsin, and take 10 microL to mix with the same volume trypan blue, and finally counting. Once this point has been verified, it would go on to check other experimental conditions.

María Luisa Caballero thank you very much for your suggestion. I had 7 repeats of each condition. Only some of them turned too blue. If I had paralel samples - one for counting the cells and one for developing the MTT to formazan, I could end up counting the "normal" well. However, I really appreciate your suggestion and I will think about it, if I can involve it in the next experiment.

Good luck!

Hi Terezie,

From my understanding, the well you expected to see less viability still shows blue color, which could refer to false-positive viability. MTT assay can be affected by your treatment if it has a reducing activity, especially when they reduce the MTT at the same location as NADH/NADPH dehydrogenase does. The classic example is that some plant extracts did it that way (https://pubmed.ncbi.nlm.nih.gov/30814856/).

However, you mentioned just the insulin (which I don't think could reduce the MTT) in your experiment. I don't know what are the specific conditions in your experiment, but you should consider if is there anything that has the reducing power in some specific conditions (blue color wells).

If you want to compare with other assays that use different principles, you can take a look. Such as SRB assay, ATP assay, LDH leakage assay, and/or neutral red uptake assay.

Dear Karnchanok Kaimuangpak , thank you for your response. I indeed think they are false positives. I treated all cells with hydrogen peroxide (oxidative agent), except for control. I will definitely read the paper you suggested.

I had also observed same thing when my experiment got contaminated with some microbes.