Caspase 3 fitc assay kit

To assess the effect of arsenic trioxide on late apoptosis, caspase-3 assay was performed by flow cytometry according to previously described protocols [34 (link)–36 (link)] using a commercially available caspase-3 FITC assay kit (BD Pharmingen). A549 cells were seeded at a density of 3 × 10⁵ cells into F12-K complete medium on 13 × 100 mm tissue treated plate and grown to 60–70% confluence in 3 days. Sub-confluent cells were serum starved overnight. The cells were re-introduced to F12-K complete medium and treated with arsenic trioxide at 0, 2, 4, and 6 µg/ml, respectively for 48 hr. After exposure, cells were washed twice with cold PBS and resuspended in BD Cytofix/Cytoperm (neutral pH-buffed saline, saponin and 4% (w/v) paraformaldehyde) at a concentration of 1 × 10⁶ cells/ 0.5 ml. The cells were incubated for 20 min on ice. The cells were pelleted and the BD Cytofix/Cytoperm solution was aspirated and discarded. The cells were washed twice with BD Perm/Wash buffer at room temperature. The cells were resuspended in BD Perm/Wash buffer plus antibody and incubated for 30 min at room temperature. The pellets were resuspended in BD Perm/Wash buffer and analyzed and counted at 10,000 counts on the fluorescence-activated cell-sorting (FACS-Vantage) system (Becton-Dickinson, San Jose, CA).