Establishment of a flow cytometry-based assay for the detection of monocyte-mediated antibody-dependent cell-mediated cytotoxicity

Abstract

Objective To establish a flow cytometry-based assay for the detection of monocyte-mediated antibody-dependent cell-mediated cytotoxicity (ADCC). Methods P815 cells double stained with PKH26 and carboxyfluorescein succinimidyl ester (CFSE) were used as target cells and coated with P815 specific antibodies to form antigen-antibody complexes. The peripheral blood mononuclear cells were isolated as effector cells and co-cultured with the antigen-antibody complexes. The CD3-CD14+ PKH26+ CFSE- cell population were gated by flow cytometry. Optimized effector/target cell ratio and incubation time for killing assay were identified. Monocyte-mediated ADCC in 23 patients with chronic HCV infection and 22 healthy subjects were analyzed. Results The monocyte-mediated ADCC could be evaluated through analyzing the CD3-CD14+ PKH26+ CFSE- cells with flow cytometry. The optimized effector/target cell ratio was 10∶1 and the optimized time for incubation was 4 h. Monocyte-mediated ADCC was inhibited in patients with chronic HCV infection as compared with healthy subjects (P=0.009). Conclusion A flow cytometry-based assay for the detection of monocyte-mediated ADCC was established, which could be used as a fast, sensitive and safety method for the evaluation of monocyte-mediated ADCC during viral infections and the research and development of drugs. Key words: Monocytes; Antibody-dependent cell-mediated cytotoxicity; Flow cytometry