Comparative study of four fluorescent probes for evaluation of natural killer cell cytotoxicity assays

Cytotoxicity is one of the major defence mechanisms against both virus-infected and tumor cells. Radioactive (51)chromium (Cr-51) release assay is a "gold standard" For assessment of natural killer (NK) cytolytic activity in vitro. Several disadvantages of this assay led LIS to design alternative tools based oil flow cytometry analysis. Four different fluorescent dyes, calcein acetoxymethyl ester (CAM), carboxyfluorescein succinimidyl ester (CFSE), Vybrant DiO (DiO) and MitoTracker Green (MTG) were tested for labeling of NK target K-562 cells. Target staining stability, Spontaneous release Of fluorochromes and Subsequent accumulation in bystander unstained cells were measured using fluorimetry and flow cytometry. Healthy donor peripheral blood mononuclear cells and affinity column Purified NK Cells Were used Lis effectors coincubated with target K-562 cells at different E:T ratios for 3 h a rid respectively. Fluorescent probe 7-amino-actinomycin D was used for live and dead cell discrimination. Bland-Altman statistical method was applied to measure true agreement for all CAM-Cr-51, CFSE-Cr-51, DiO-Cr-51 and MTG-Cr-51 pairs analyzed. Based on the data, none of the four proposed methods call be stated equivalent to the standard Cr-51 release assay. Considering linear relationships between data obtained with four fluorochromes and 5 1 Cl, release assay as well as linear regression analysis with R-2 = 0.9393 value for CAM-Cr-51 pair, we Found the CAM assay to be the most closely related to the Cr-51 assay. (C) 2008 Elsevier GmbH. All rights reserved.