Abstract

This research objective was to exploit a novel method for measuring the proliferation, cytotoxicity of cytokine-induced killer (CIK) cells using carboxyfluorescein succinimidyl ester/proliferation index (CFSE/PI) and flow cytometric assay. As cells divide, CFSE is apportioned equally between the two daughter cells, leading to a characteristic flow cytometric profile where a number of peaks of progressively halving CFSE fluorescence intensity were observed. The test principle of cytotoxicity is based on target cell labeling with CFSE and subsequent DNA-labeling with PI for the identification of target cells with compromised cell membranes. Results of CFSE fluorescence profile of the entire cell population showed that these cells underwent up to 6 divisions after 21 days. The percentage of total dividing cell population was 97.12%, of which 17.28% cells went through 6 rounds and 6.84% cells entered the seventh generation. The cytotoxicity of CIK cells showed a significant and positive correlation with effector: target (E:T) ratio ranging from 50:1 to 6.25:1. CFSE labeling combined with flow cytometry can therefore be used to monitor the proliferation of CIK cells. Moreover, it presents an advantageous method to measure cytotoxicity of CIK cells by avoiding radioactive substance, increasing sensitivity at low E:T ratio, analyzing on a cell-to-cell basis and allowing for long-term incubation.

Key words: Flow cytometry, CFSE, cytokine-induced killer cell, proliferation, cytotoxicity.

Abbreviation

CIK, Cytokine-induced killer; PBMCs, peripheral blood mononuclear cells; IL-2, interleukin-2; IL-1α, interleukin-1; IFNγ, interferon-gamma;anti-CD3mAb, monoclonal antibody against CD3; CTL, cytotoxic T lymphocytes;CFDA-SE, carboxyfluorescein diacetate succinimidyl ester; IMDM, Iscove’s modified Dulbecco’s medium; FCS, fetal calf serum; CM, complete medium; FBS,fetal bovine serum; PI, proliferation index; CFSE, carboxyfluorescein succinimidyl ester; LDH, lactate dehydrogenase.