Quantitative fluorescence cytometric analysis of Bcl-2 levels in tumor cells exhibiting a wide range of inherent Bcl-2 protein expression: Correlation with Western blot analysis

Background: A protocol to measure a wide range of Bcl-2 protein expression using quantitative fluorescence cytometry (QFCM) in different cell types wa s developed for use with now cytometry. Bcl-2 measurements obtained by flow cytometry were correlated with Western blot Bcl-2 measurements to confirm specificity of the Bcl-2-FITC staining. This protocol was applied to measur e absolute levels of Bcl-2 protein in different tumor cell Lines including Bcl-2-transfected breast carcinoma cell lines and in peripheral blood lymph ocytes (PBL). Methods: HL-60, K562, DOHH2, Jurkat, MDA435/LCC6, MCF7 cell Lines, and PBL derived from normal donors were fixed, permeabilized, stained with anti-Bcl -2-FITC antibody and evaluated by QFCM. In parallel, the same cells were ev aluated for Bcl-2 protein expression by Western blot analysis. Mitochondria l localization of anti-Bcl-2-FITC antibody inside cells was confirmed using fluorescence imaging microscopy. Results: Bcl-2 expression in different cell types could be accurately quant ified based on antibody-binding capacity (ABC) ranging from 12.6 X 10(3) an tibody-binding sites in HL-60 cells to 1.64 X 10(6) antibody binding sites in a Bcl-2-transfected MDA435/LCC6 clone. The data from flow cytometry anal ysis correlated well with Western analysis (R-2 = 0.78). Bcl-2-FITC stainin g colocalized with dyes specific for mitochondria. Conclusions: The Bcl-2 staining protocol described here was shown to be spe cific, sensitive, and it was able to provide higher resolution as well as m ore reproducible quantitation of Bcl-2 protein content in cells when compar ed with Western blot methods. Quantitation of Bcl-2 content in cells by QFC M may be useful for monitoring Bcl-2 expression in cells undergoing various treatments in vitro and in vivo. (C) 2000 Wiley-Liss, Inc.