Immunocytofluorescent Analysis of HBEC2 Cells

Immunocytofluorescent analysis was performed as previously described [52 (link)]. Twenty-four hours after culture in the presence or absence of 50 μM Apo9F on Lab-Tek-II 8-chamber slides (Nalge Nunc International, Rochester, NY, USA), HBEC2 cells were further cultured with or without 1.5% CSE for another 24 h. Cells were fixed with 2% (w/v) paraformaldehyde in PBS for 15 min at 37 °C. The cells were then incubated with 0.2% Triton X-100 with 0.2% Saponin in a blocking solution containing 3% IgG-free BSA (bovine serum albumin), 1% Gelatin and 2% normal donkey serum for 1 h at RT (room temperature) and further incubated with the following primary antibodies at 4 °C overnight: phosphorylation-specific antibody for ATM. The immunolabeled cells were detected using F(ab)2-fragments of respective secondary antibody conjugated to DylightTM-549 (Jackson ImmunoResearch, West Grove, PA, USA) and mounted with 4′,6-diamidino-2-phenylindole (DAPI) containing Fluormount-G™ (SouthernBiotech, Birmingham, AL, USA) for nuclear staining.