XTT Assay: A Colorimetric Assay to Assess Cell Viability and Mitochondrial Activity

- Low-frequency ultrasound waves lead to the selective lysis of human leukemia cells and the XTT assay helps determine the degree of this damage. The assay works on the principle that the mitochondrial enzymes of healthy cells convert a yellow XTT dye into an orange compound called formazan. To begin the assay, treat the cells with methyl beta-cyclodextrin, a cholesterol-depleting agent, and subsequently sonicate the cells at low power to induce cell damage.

Centrifuge to pelletize the cells and add fresh cell growth media to the pellet. Seed the cells at the desired concentration into a multi-well microplate and incubate it overnight. Now, add the activated XTT solution to the cells and incubate until the orange color develops in the microplate's wells. Place the plate in a spectrophotometer. Measure the absorbance at 450 to 500 nanometers and then at 630 to 690 nanometers. Subtract the two values to get precise results. The higher value of absorbance in the XTT assay indicates the higher activity of mitochondrial enzymes and higher cell viability.

In the following video, we will demonstrate the use of XTT assay for determining the efficacy of low-frequency ultrasound waves to cau