Thaw out Resazurin solution (if kept frozen) and warm it to 37°C to ensure all components are completely in solution.
Plate cells in 96-well plate (black plate with clear bottom). Typical seed cell number is 0.02–2 x 105 cells/well depending on cell growth rate. Cell number titration is recommended to determine the optimal cell seeding density.
Incubate cells with compound of interest for desired period of time (1–72 hr). Make sure all the wells contain the same volume of medium.
Add Resazurin solution to plate (10% of the initial volume in the well). For example, for plates containing 100 μl medium/well, add 10 μl resazurin solution to each well.
Incubate the plate for 1–6 hr in standard culture conditions. Incubation time depends on cell type and cell number. The plate can be read multiple times to determine the optimal time point.
Measure the relative fluorescent units (RFU) using a plate reader. Ex = 530–570 nm, Em = 590–620 nm.
