Annexin 5 propidium iodide assay

The annexin V/propidium iodide assay was performed according to the manufacturer’s instructions (Invitrogen, Carlsbad, California, USA) to analyze apoptosis. NOZ and SGC-996 cell lines were seeded into 6-well plates (Corning) with 1 × 10⁶ cells per well and treated with osthole (0, 50, 100, and 150 μM). After incubated for 48 hours, the cells were collected and washed twice with cold PBS, then centrifuged and resuspended at a density of 1 × 10⁶ cells/ml into 100 μl of binding buffer containing 5 μl of Annexin V-FITC and 5 μl of PI working solution (100 μg/ml). After incubated at room temperature for 15 minutes in the dark, 100 μl of binding buffer was added to each sample. The stained cells were analyzed by flow cytometry (BD, San Diego, California, USA) for at least 10 000 events. Cells populations in different quadrants were measured by quadrant statistics.

The detection of apoptosis by the annexin-V–propidium iodide assay (Invitrogen) and TUNEL assay (AbD Serotec, Oxford, UK) was performed as previously described.²⁸ For ER stress agents, an appropriate dose was selected from a range of concentrations tested. BFA (Sigma), TG (Sigma) and TM (Sigma) were used at 10 μg/ml.