Alamar Blue assay optimization to minimize drug interference and inter assay viability

Alamar Blue (AB) has become an increasingly popular reagent of choice for cell viability assays. We chose AB over other reagents such as MTT and Cell-Titer Glo due to its cost effectiveness and its ability to be a nondestructive assay. While analyzing the effect of osimertinib, an EGFR inhibitor on the non-small cell lung cancer cell line, PC-9, we noticed unexpected right-shifts of dose response curves as compared to the curves obtained by Cell Titer Glo assay. Here, we describe our modified AB assay method to avoid that right shift in dose response curves.•Removal of the drug containing medium prior to AB addition eliminated the falsely increased readings, giving comparable dose response curves as determined by Cell Titer Glo assay.•Plate-to-plate variability can be minimized by adding an appropriate concentration of a common fluorescence calibration standard to the assay plates to calibrate fluorimeter sensitivity.•Alamar Blue can be used as a continuous longitudinal assay to monitor cell growth or recovery from drug toxicity over time.