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 was developed for use with flow 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 measure absolute levels of Bcl-2 protein in different tumor cell lines including Bcl-2-transfected breast carcinoma cell lines and in peripheral blood lymphocytes (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 evaluated for Bcl-2 protein expression by Western blot analysis. Mitochondrial 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 quantified based on antibody-binding capacity (ABC) ranging from 12.6 x 10(3) antibody-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 analysis correlated well with Western analysis (R(2) = 0.78). Bcl-2-FITC staining colocalized with dyes specific for mitochondria.


CONCLUSIONS
The Bcl-2 staining protocol described here was shown to be specific, sensitive, and it was able to provide higher resolution as well as more reproducible quantitation of Bcl-2 protein content in cells when compared with Western blot methods. Quantitation of Bcl-2 content in cells by QFCM may be useful for monitoring Bcl-2 expression in cells undergoing various treatments in vitro and in vivo.
Department of Advanced Therapeutics, BC Cancer Agency, Vancouver, Canada.
W H
WH
Dragowska
D E
DE
Lopes de Menezes
J
J
Sartor
L D
LD
Mayer
D015153:Blotting, Western / Q000379:methods; D001943:Breast Neoplasms / Q000737:chemistry* / Q000235:genetics / Q000473:pathology; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D015870:Gene Expression; D006801:Humans; D019044:Image Cytometry / Q000379:methods*; D007074:Immunoglobulin G / Q000032:analysis; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000737:chemistry; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis* / Q000235:genetics; D014162:Transfection / Q000235:genetics; D014407:Tumor Cells, Cultured
D007074:Immunoglobulin G; D019253:Proto-Oncogene Proteins c-bcl-2