A study of some variables in a tetrazolium dye (MTT) based assay for cell growth and chemosensitivity

Abstract

We have studied various factors involved in the optimal use of a tetrazolium (MTT) basedcolorimetric assay for cell growth and chemosensitivity. The assay is dependent on the ability of viable cells tometabolise a water-soluble tetrazolium salt into a water-insoluble formazan product. We have found thatDMSO is the best solvent for dissolving the formazan product, especially where a significant amount ofresidual medium is left in the wells of the microtitre tray used for the assay. A reaction occurs betweenmedium and a solution of MTT formazan in DMSO which changes the shape of the absorbance spectrum ofthe solution. The resulting optical density is not however greatly dependent upon the volume of addedmedium in the range 1-10 µl. Between 10 and 40 µl of added medium results in a gradually lower opticaldensity than that produced by the smaller volumes. Above 40 µl, the optical density increases again due toturbidity as protein precipitation occurs. When cells are incubated with MTT, the resulting optical density ofthe formazan product is dependent upon both the concentration of MTT and the incubation time. The opticaldensity is stable for several hours after solution of the formazan in DMSO. A linear relationship is seenbetween optical density and cell number for incubation times of 2, 4, 6 or 24 h with 20 µl of MTT (5mg ml-1)added to 200 µl medium. We have adopted 4 h as the standard incubation time for the assay. Only a smallamount of MTT formazan product can be detected in the growth medium of wells in which cells have beenexposed to MTT.Comparative chemosensitivity data for EMT6 mouse tumour cells show good agreement between resultsobtained using the MTT assay and results based on total cell count after a fixed period of growth. © The Macmillan Press Ltd., 1987.