The modified FACS calcein AM retention assay: A high throughput flow cytometer based method to measure cytotoxicity

Current methods to determine cellular cytotoxicity in vitro are hampered by background signals that are caused by auto-fluorescent target and effector cells and by non-specific cell death. We combined and adjusted existing cell viability assays to develop a method that allows for highly reproducible, accurate, single cell analysis by high throughput FACS, in which non-specific cell death is corrected for. In this assay the number of living, calcein AM labeled cells that are green fluorescent are quantified by adding a fixed number of unlabeled calibration beads to the analysis. Using this modified FACS calcein AM retention method, we found EC50 values to be highly reproducible and considerably lower compared to EC50 values obtained by conventional assays, displaying the high sensitivity of this assay. 1.Coat a 96 well flat bottom culture plate with Poly-d-Lysin.2.Seed adherent target cells 3days prior to the cytotoxicity assay. Seed non-adherent cells after step 3.3.Calcein labeling of target cells on the day of the assay.4.Add test antibodies or compound.5.Add complement for CDC assay (a) or effector cells for ADCC (b) (optional).6.Incubate at 37°C in the dark for 1hour (CDC) or 4hours (ADCC), then add 10,000 FACS beads.7.Acquire 5000 beads by FACS. Process data with Flowjo. [Display omitted] •The modified FACS calcein retention assay is a highly reproducible method to determine cytotoxicity.•The method quantifies live cells by measuring labeled cells relative to calibration beads.•The method can be applied to non-adherent and adherent target cells.