A novel enhanced green fluorescent protein (EGFP)-K562 flow cytometric method for measuring natural killer (NK) cell cytotoxic activity

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Abstract

Enhanced green fluorescent protein (EGFP) was stably expressed in human erythroleukaemia K562 cells (EGFP-K562) and used as target cells for measurement of natural killer (NK) cell cytotoxicity by flow cytometry. The compromised EGFP-K562 target cells were stained with propidium iodide (PI) and showed dual (green–red) fluorescent. Although the kinetic study demonstrated that the optimal incubation time for the assay was 4 h, a 2-h incubation period also gave comparable results. This new technique correlated strongly with the standard chromium (51Cr) release assay at the correlation coefficients of 0.87 and 0.89 at p-value <0.001 for 2- and 4-h incubation times, respectively. The EGFP-K562 stable cell line provides a novel method to measure NK cytotoxicity by flow cytometry without pre-staining or pre-labeling target cells.

Introduction

Natural killer (NK) cells are involved in innate immunity. They kill various tumor cell- and viral infected cell-targets. The standard chromium (51Cr) release assay using the human erythroleukaemia K562 cell line as target cells has been the method of choice for studying NK activity in vitro since 1968 (Brunner et al., 1968). However, many unfavorable characteristics of chromium still exist including a short half-life, high cost, health risk, and spontaneous release of 51Cr from target cells Slezak and Horan, 1989, Goldberg et al., 1999. A number of alternative methods have been proposed to evaluate NK activity. These include enzymatic assays (Korzenniewski and Callewaert, 1983) and fluorescent dye marker assays Blomberg et al., 1986, Kolber et al., 1988, Volgmann et al., 1989, Wierda et al., 1989, Kroesen et al., 1992. Moreover, many different fluorescent dyes have been used to stain targets for measuring NK activity by flow cytometry Legendre et al., 1986, McGinnes et al., 1986, Shi et al., 1987, Callewaert et al., 1991, Radcliff et al., 1991, Chang et al., 1993, Papadopoulos et al., 1994, Johann et al., 1995. However, it is hard to control the quality of the pre-stained targets. In addition, the process of staining may alter target susceptibility to lysis. Some dyes exhibit a low intensity of fluorescent signal (Kroesen et al., 1992) while others have shown high spontaneous leakage (Kolber et al., 1988).

The green fluorescent protein (GFP) of jellyfish, Aequorea victoria, was modified to give a higher fluorescence emission as enhanced GFP (EGFP) (Bierhuizen et al., 1997). EGFP has now become a very popular reporter gene. It works well with flow cytometry (Van Tendeloo et al., 2000) and fluorescent microscopy (Li et al., 1998). In this study, we developed an alternative simple, rapid and reproducible NK cytotoxicity assay using an EGFP-transfected K562 stable cell line and flow cytometry.

Section snippets

Cell line

A human erythroleukaemic cell line, K562, (American Type Culture Collection no. CCl-243) was cultured in growth medium containing RPMI 1640 (Gibco, Grand island, NY, USA) supplemented with 2 mM l-glutamine, 100 μg/ml streptomycin, 100 IU/ml penicillin and 10% heat inactivated fetal calf serum (FCS) (Gibco) at 37 °C with 5% CO2. When antibiotic selection was applied, cells were grown in growth medium with 400 μg/ml neomycin analogue G418 (Bio-Rad, Hercules, CA, USA). One day prior to the assay,

Establishment of EGFP-K562 stable cell line

All K562 transfectant cells were viewed using an inverted fluorescence microscope from 2 days after transfection. Seven days after transfection, those cells that showed expression of EGFP were further selected by limiting dilution using G418. At the end of the first month, 12 clones of EGFP-K562 stable cell line with various levels of fluorescence emission were established. A clone, JJ111, showed the highest intensity emission of EGFP. The level of fluorescence analyzed by flow cytometry was

Discussion

NK cells play a critical role in nonspecific host defense mechanisms. They kill tumor cells and viral infected cells. The 51Cr release assay has been a standard method to study NK cell activity (Brunner et al., 1968). However, the constraints of using 51Cr have convinced several investigators to develop alternative approaches. The most popular methods have been based on two-color flow cytometry. Target cells were labeled with a fluorescent dye before use in the assay, and a second fluorescent

Acknowledgements

We are grateful to Mr. Suthipol Udompunturuk for the statistical analysis and Dr. Mark de Souza for the critical reading of the manuscript. This work was supported by a grant from the Ministry of University Affairs 1999–2001.

References (26)

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