FlowCytometricAnalysisOfBclFamilymembers

Description
Cell Fixation, staining and flow cytometric analysis 

Procedure
Cells (106) were washed twice in FACS buffer (phosphate buffered saline PBS pH 7.4 containing 2% fetal calf serum(FCS) and 0.02% NaN3) and resuspended in 250ƒY´l chilled fixation buffer (1% paraformaldehyde in FACS buffer) and incubated on ice for 10 minutes, followed by addition of 30ƒY´l 0.5% Tween 20 and further incubated for 8 minutes. The cells were washed twice in FaCS Buffer and resuspended in FCS for 30 minutes to prevent non-specific antibody binding. Cells were washed thrice in FACS buffer, incubated with the relevant antibody or relevant isotype matched control antibodies at 4„aC for 40 minutes. This was followed by three washes in FACS buffer and incubation for 30 minutes at 4„aC with the secondary antibody. The cells were washed thrice and analyzed. Single color flow cytometry was performed on FACSCALIBUR (Becton Dickinson Immunocytometry Systems), equipped with 488 nm argon laser and data was analyzed using CELLQUEST software. Live cells were gated on the basis of forward and side scatter and a minimum of 10,000 events were analyzed. 

Recipes
The primary antibodies used were mouse monoclonal AB against Bcl-2 (Dako, USA), rabbit polyclonal against Bcl-xL (Santacruz Biotechnology, USA) and rabbit polyclonal against Bax (Santacruz Biotechnology, USA). The secondary AB against Bcl2 was goat anti mouse Ig FITC (Immunotech, USA) and against Bcl-xL and Bax goat anti rabbit FITC (ICN-Cappel, USA). FITC isotype specific monoclonal ABs (Sigma, USA) of irrelevant specificities served as negative controls.