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 CTL and NK cell-mediated cytotoxicity. After effector/target cells are incubated together, CTL or NK populations are stained with an effector cell specific PE-conjugated mAb. Subsequently, annexin V-FITC binds to cells expressing phosphatidylserine (an early marker of apoptosis) on the cell surface. Target cells are gated upon as PE-negative and quantified with respect to their annexin V positivity. The shift from annexin Vneg to annexin Vhi is a discrete event such that all target cells fall within discernible populations with respect to annexin V. There is a strong correlation between cytotoxicity measured with our assay and a standard 51Cr release assay (r2 = 0.989). The PE/annexin V assay shows increased sensitivity at early timepoints after target/effector cell mixing. In addition, this method allows for analysis of target cells at the single cell level. Therefore, we have described a promising new technique to measure in vitro cell-mediated cytotoxicity. It avoids the potential difficulties of working with radioactive isotopes, and offers increased sensitivity and versatility.
Goldberg
J E
JE
Department of Cardiothoracic Surgery, Stanford University School of Medicine, CA 94305-5407, USA.
Sherwood
S W
SW
Clayberger
C
C
D017304:Annexin A5 / Q000032:analysis*; D015415:Biomarkers; D016827:CD8 Antigens; D002460:Cell Line; D002857:Chromium; D003601:Cytotoxicity Tests, Immunologic / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006801:Humans; D019169:Jurkat Cells; D007694:Killer Cells, Natural / Q000276:immunology*; D010799:Phycoerythrin; D013194:Staining and Labeling / Q000379:methods; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
D017304:Annexin A5; D015415:Biomarkers; D016827:CD8 Antigens; D005456:Fluorescent Dyes; D002857:Chromium; D010799:Phycoerythrin; D016650:Fluorescein-5-isothiocyanate