Apoptosis assay

Flow cytometric analysis was used to detect apoptosis by examining altered plasma membrane phospholipid packing by the lipophilic dye Annexin V. Briefly, cells were treated with Gef and/or Met for 48 h, harvested by trypsin, washed twice with PBS and then resuspended at a density of 1 × 10⁷ cells/mL. Thereafter, 5 μL of Annexin V-FITC and 5 μL of propidium iodide (PI) were added to 100 μL of the cell suspension and incubated for 30 min at room temperature in the dark. Next, labeled cells were processed by flow cytometry. All early apoptotic cells (i.e., Annexin V positive, PI negative), necrotic/late apoptotic cells (i.e., double positive) and living cells (i.e., double negative) were detected by using a Cytomics FC 500 flow cytometer (Beckman Coulter, Miami, FL, USA).