Annexin 5 pi staining assay

Annexin-V/PI staining assay (BD Biosciences, San Jose, CA) was performed as previously described^{32 (link)}. HN3 cells were plated at 1 × 10⁶ cells/well in 6-well plates and were treated with TRAIL and/or VPA. After 24 h of cell seeding, the apoptotic cells were stained with Annexin-V-FITC and PI solution, and the stained cells were analyzed via flow cytometry (BD Biosciences).

Panc1 and ASPC1 cells were seeded at a density of 200,000 cells/well in a 12-well plate under standard conditions and treated with chemicals as indicated in the text. After 24 h incubation cells were trypsinized and collected by centrifugation. The annexin v/PI staining assay (BD, Germany) was used to analyze cell apoptosis according to the manufacturer’s protocol. Briefly, the cells were re-suspended in the binding buffer and stained with FITC-conjugated annexin v for 15 min, followed by PI staining for 5 min at room temperature. The cell suspension was immediately analyzed by FACS. An aliquot of the re-suspended cells was used to determine the cell number after treatment, using a hemacytometer and trypan blue. The relative amount of cells was calculated as the number of trypan blue negative cells divided by the number in the mock treatment.