Resazurin Live Cell Assay: Setup and Fine-Tuning for Reliable Cytotoxicity Results

In vitro cytotoxicity tests allow for fast and inexpensive screening of drug efficacy prior to in vivo studies. The resazurin assay (commercialized as Alamar Blue^®) has been extensively utilized for this purpose in 2D and 3D cell cultures, and high-throughput screening. However, improper or lack of assay validation can generate unreliable results and limit reproducibility. Herein, we report a detailed protocol for the optimization of the resazurin assay to determine relevant analytical (limits of detection, quantification, and linear range) and biological (growth kinetics) parameters, and, thus, provide accurate cytotoxicity results. Fine-tuning of the resazurin assay will allow accurate and fast quantification of cytotoxicity for drug discovery. Unlike more complicated methods (e.g., mass spectrometry), this assay utilizes fluorescence spectroscopy and, thus, provides a less costly alternative to observe changes in the reductase proteome of the cells.