Hi Jose,

First, if adherent cells, make sure the cells are formed in a monolayer evenly. Uneven cell distribution will cause deviation among wells; if suspended cells, make sure to resuspend those cells thoroughly and seed the same number of cells.

Second, be very careful not to get cells away when washing wells or removing the MTT. If suspended cells, I don't think you need to remove MTT, because this step will defintely loss cells, and lead to big differences among wells.

in addition to above suggestions.

you should not consider that 20000 cells /well will give you the absorbance of 1 or similar. just seed enough number of cells which can be almost 90% confluent at the endpoint.

mix well during cell seeding even though you are using multi-channel pipette.

How can I get good reproducibility in my MTT?

Hello,

Its just handing that will give you the reproducible results.....as others mentioned.....count your cells well....seed them properly.....and avoid cell loss before MTT procedure...

Regards

Maybe use a higher concentration of MTT? Do you wash before DMSO or do you subtract OD650 (for not reacted MTT)?

Hello again,

They are adherent cells so I can remove by aspirating the media, taking care of not removing any cells. I have thought of trying reverse pipetting but i don't know if that will have some influence in the results. So, should I wash the wells after removing MTT (before adding DMSO)? with PBS for example? Wouldn't that remove MTT or cells attached?

Thank you all for your answers

You should be able to wash the adherent cells, PBS is fine. If not, subtract the signal at 650 nm.

MTT has been processed intracellularly to water insoluble formazan. DMSO dissolves formazan and destroys your cells. You can also use alamar blue (resazurin based assay) which is water soluble and is excreted into the medium.

Thank you very much for your suggestions, I will take them into account. I am opened as well to other new ideas.

Javier