title: The modified FACS calcein AM retention assay: A high throughput flow cytometer based method to measure cytotoxicity.
abstract: 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.
authors.affiliation: AIMM Therapeutics, Academic Medical Center, Amsterdam, The Netherlands; Department of Hematology, Academic Medical Center, Amsterdam, The Netherlands.
authors.forename: M A
authors.initials: MA
authors.lastname: Gillissen
authors.affiliation: AIMM Therapeutics, Academic Medical Center, Amsterdam, The Netherlands.
authors.forename: E
authors.initials: E
authors.lastname: Yasuda
authors.affiliation: AIMM Therapeutics, Academic Medical Center, Amsterdam, The Netherlands; Department of Hematology, Academic Medical Center, Amsterdam, The Netherlands.
authors.forename: G
authors.initials: G
authors.lastname: de Jong
authors.affiliation: AIMM Therapeutics, Academic Medical Center, Amsterdam, The Netherlands.
authors.forename: S E
authors.initials: SE
authors.lastname: Levie
authors.affiliation: AIMM Therapeutics, Academic Medical Center, Amsterdam, The Netherlands.
authors.forename: D
authors.initials: D
authors.lastname: Go
authors.affiliation: AIMM Therapeutics, Academic Medical Center, Amsterdam, The Netherlands.
authors.forename: H
authors.initials: H
authors.lastname: Spits
authors.affiliation: AIMM Therapeutics, Academic Medical Center, Amsterdam, The Netherlands.
authors.forename: P M
authors.initials: PM
authors.lastname: van Helden
authors.affiliation: Department of Hematology, Academic Medical Center, Amsterdam, The Netherlands. Electronic address: m.d.hazenberg@amc.nl.
authors.forename: M D
authors.initials: MD
authors.lastname: Hazenberg
mesh_terms: D001681:Biological Assay; D045744:Cell Line, Tumor; D002470:Cell Survival; D003602:Cytotoxicity, Immunologic; D005434:Flow Cytometry / Q000379:methods*; D005452:Fluoresceins / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D007694:Killer Cells, Natural / Q000276:immunology*; D000069283:Rituximab / Q000737:chemistry; D000068878:Trastuzumab / Q000737:chemistry; D014407:Tumor Cells, Cultured
chemical_list: D005452:Fluoresceins; D005456:Fluorescent Dyes; C085925:calcein AM; D000069283:Rituximab; D000068878:Trastuzumab
keywords: ADCC; CDC; Cancer; Cellular cytotoxicity; Monoclonal antibodies