A novel method for measuring CTL and NK cell-mediated cytotoxicity using annexin V and two-color flow cytometry

An assay based on two-color flow cytometry has been developed to measure CT L and Ng cell-mediated cytotoxicity. After effector/target cells are incuba ted together, CTL or NK populations are stained with an effector cell speci fic PE-conjugated mAb. Subsequently, annexin V-FITC binds to cells expressi ng phosphatidylserine tan early marker of apoptosis) on the cell surface. T arget cells are gated upon as PE-negative and quantified with respect to th eir annexin V positivity. The shift from annexin V-neg to annexin V-hi is a discrete event such that all target cells fall within discernible populati ons with respect to annexin V. There is a strong correlation between cytoto xicity measured with our assay and a standard. Cr-51 release assay (r(2) = 0.989). The PE/annexin V assay shows increased sensitivity at early timepoi nts after target/effector cell mixing. In addition, this method allows for analysis of target cells at the single cell level. Therefore, we have descr ibed a promising new technique to measure in vitro cell-mediated cytotoxici ty. It avoids the potential difficulties of working with radioactive isotop es, and offers increased sensitivity and versatility. (C) 1999 Elsevier Sci ence B.V. All rights reserved.