MTT Assay

Hi everyone, gwen here. I am going to share with you about MTT assay. This assay is done to determine the potency of the drug and proteins used for cancer therapy. Each treatment can consist of the drug with the protein or just the drug or protein alone.

The first step to this assay would be to seed the desired cells into 96-well plates. The number of plates needed would depend on the number of treatments planned. The cells would then be incubated and treated with treatments the next day. Each plate will have 12 columns altogether. 2 columns are reserved as controls and the cells in the remaining 10 columns of cells will be treated with the treatments.

After overnight incubation, the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) solution is added to the cells after removing the used media/treatments. The plates are then incubated for another 3-5 hours. During this time, the MTT forms purple formazan from the living cells. After the incubation, the remaining MTT solution would be removed. Care must be taken so as not to pipette the purple formazan out, as it will affect the viability results.DMSO is then added to each well of the plate so as to dissolve the purple formazon, then the absorbance level can be read and the viability of the cells determined. Viability is calculated by dividing the absorbance level of the treated cells with that of the control and a percentage is derived.