QUANTIFICATION OF MITOGEN-INDUCED HUMAN LYMPHOCYTE-PROLIFERATION - COMPARISON OF ALAMARBLUE(TM) ASSAY TO H-3 THYMIDINE INCORPORATION ASSAY

Proliferation of human lymphocytes in response to various stimuli has traditionally been assessed by measuring uptake of radiolabeled nucleo tides such as H-3-thymidine. We have evaluated a fluorometric assay, w hich uses the commercially available reagent, alamarBlue, as a potenti al substitute for the H-3-thymidine assay in measuring proliferation o f human lymphocytes. In this assay, alamarBlue(TM) is added to a popul ation of cells where it is reduced by mitochondrial enzyme activity. T he reduced form of the reagent is fluorescent and can be quantitativel y detected. The safety and convenience of the alamarBlue(TM) assay mak e it very attractive for use in the clinical laboratory. In this study peripheral blood mononuclear cells (PBMC) from healthy donors were st imulated using the mitogen Concanavalin A, and proliferation was asses sed using either the H-3-thymidine or the alamarBlue(TM) assay. The al amarBlue(TM) assay reliably detects human PBMC and we found that the l inear range of detection was 10(4) cells/well (96-well plate) to 5 x 1 0(5) cells/well. Detection of human PBMC is highly reproducible and th e alamarBlue assay may be suitable in a number of applications where d etection or relative quantitation of human PBMC is required. The alama rBlue assay also detected mitogen induced proliferation of PBMC althou gh with a significantly lower lever of sensitivity than the standard H -3-thymidine assay. (C) 1995 Wiley-Liss, Inc.