2.5. Assessment of pro‐apoptotic conformational changes of Bak and Bax

Bak and Bax activation was measured by flow cytometry using antibodies recognizing N‐terminal conformational changes previously shown to be associated with pro‐apoptotic function of either protein (Griffiths et al., 1999; Mandic et al., 2002). J82 cells were after treatment fixed in 4% paraformaldehyde solution in PBS for 15 min and stained with antibodies against Bak (AM03, clone TC100; Oncogene Research Products, CA, USA), 1:50, Bax (clone 6A7; BD Biosciences Pharmingen, San Diego, CA, USA), 1:250 in 100 μl of PBS containing digitonin (100 μg/ml) as permeabilising agent for 1 h. A secondary Alexa 488 labelled anti‐mouse antibody was applied for 45 min after which Bak or Bax associated IFL signal was recorded in the FL‐1 channel of the FACS Calibur flow cytometer (BD Bioscience). Cell Quest software (BD Bioscience) was used to process the data. The accurate staining with Bak or Bax antibodies was verified using an isotype IgG1 negative control antibody (data not shown). The fold change, in Bak or Bax associated median fluorescence intensity, was compared to untreated cells and mean ± SD from three replicates are presented.