Establishment of CFSE labeling method coupling with flow cytometry used in the study of human NK cell function

FAN Yanying; WU Changyou

Abstract：To establish an optimal CFSE labeling methods for the study of NK cell functions.We purified human NK cells and la-beled the cells with CFSE; the labeled NK cells were cultured in presence or absence of interleukin 2 (IL-2 ) at 37℃5% CO_2 incubator, and then harvested at day 3, 5 and 7 for determining the proliferation and divisions of activated NK cells by using flow cytometry.For analy-sis of cytolytic activity, the NK cells were incubated with IL-2 and co-cultured with target cells for 4 h at 37℃ 5% CO_2 incubator, and then propidium iodide (PI) was added into the cultures.CFSE~+PI~+ cells were determined as the dead cells by flow cytometry.The proliferation of IL-2-activated NK cells could be detected at day 5, and about 80% of NK cells were on proliferation at day 7 and divided from one to six times.On the other hand, the cytolytic effects of NK cells on CFSE-labeled different target cells were enhanced after activation with IL-2 and IL-12.The results showed that the fluorescent label ester CFSE is a convenient method to tag proliferating cells, which allows discrimi-nation of successive rounds of cell division.Based on target cells labeled with CFSE and subsequent DNA labeled with PI, the cytotoxicity of NK cells could be detected in a single cell level.This study might provide valuable information on the function study methods for NK cells as well as other lymphocyte subsets.