MTT cell viability Protocol

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Guy Sovak
Guy Sovak's picture
MTT cell viability Protocol

MTT Assay of Cell Viability Protocol
MTT: Thiazolyl Blue Tetrazolium Bromide

1) Plate 10000 cells in 150ul media per well in a 96 well plate. Leave some wells empty ( blank).
2) Incubate (37C, 5% CO 2) Over night
3) Make 5ml of MTT solution 5mg/ml in PBS. MTT should be freshly made.
4) Add 20ul MTT solution to each well. Place on a shaking table, for 5 minutes, in order to mix the MTT into the media.
5) Incubate (37C, 5% CO 2) for 3-4 hours (MTT to be metabolized).
6) Discard the media and dry the wells.
7) Resuspend formazan (MTT metabolic product) in 200ul DMSO. Place on a shaking table, 5 minutes, to thoroughly mix the formazan into the solvent.
8) Read optical density at 560nm and subtract background at 670nm. Check blank Optical density should be directly correlated with cell quantity.
*Could also be used to check effet of drugs on the cells, in that case incubate the drug with the cells after step 2 for the same time as you are dong in your experiments use the apropriate control(solvents).

madelingirly's picture

Your protocol is very helpful, however, we have baught a new microplate reader, specifically iMark bio-rad. and it has no standard curve for MTT, so please help me how I can construct these data???