2.4 |. Annexin V apoptosis assay

Apoptosis induction in vehicle- or inhibitor-treated cells was assayed by the detection of membrane externalization of phosphatidylserine using an Annexin V assay kit (Molecular Probes, Invitrogen, Carlsbad, CA) as described previously.²⁴ 2 × 10⁵ cells were harvested at various intervals after treatment, washed with ice-cold phosphate-buffered saline (PBS), and resuspended in 200 μL of binding buffer. Annexin V-FITC and 1 μg/mL propidium iodide were added and cells were incubated for 15 min in a dark environment. Labeling was analyzed by flow cytometry with a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). Annexin V binds to phosphatidylserine, which trans-locates from the inner leaflet to the outer leaflet of the plasma membrane in apoptotic cells, so cells that are positive for annexin V staining (ie, high Annexin V signal) are undergoing apoptosis. PI staining provides a measure of cell viability and is used to distinguish between cells in early and late apoptosis.