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

Cytometry. 2000 Aug 1;40(4):346-52. doi: 10.1002/1097-0320(20000801)40:4<346::aid-cyto10>3.0.co;2-w.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blotting, Western / methods
  • Breast Neoplasms / chemistry*
  • Breast Neoplasms / genetics
  • Breast Neoplasms / pathology
  • Female
  • Flow Cytometry / methods
  • Gene Expression
  • Humans
  • Image Cytometry / methods*
  • Immunoglobulin G / analysis
  • Microscopy, Fluorescence
  • Mitochondria / chemistry
  • Proto-Oncogene Proteins c-bcl-2 / analysis*
  • Proto-Oncogene Proteins c-bcl-2 / genetics
  • Transfection / genetics
  • Tumor Cells, Cultured

Substances

  • Immunoglobulin G
  • Proto-Oncogene Proteins c-bcl-2