Apoptosis measurement

Phosphatidylserine externalization was quantified by flow cytometry by using the Annexin V-FITC Apoptosis Detection Kit including propidium iodide (PI) according to the manufacturer’s guideline (Santa Cruz, CA, USA). Briefly, both attached and floating cells were collected after treatment(s), washed twice with cold PBS and re-suspended in the annexin-binding buffer at a concentration of ~1×10⁶ cells/ml; 100 μl of the cell suspension (~1×10⁵ cells) were transferred to a culture tube and 2.5 μl of annexin V-FITC and 10 μl of PI were added. After incubation in the dark (15 min at room temperature), 400 μl of the binding buffer were added and cells were analyzed immediately by flow cytometry with the DAKO Galaxy flow-cytometer equipped with HBO mercury lamp. Analysis by flow cytometry used the FL1 (FITC) and FL3 (PI) laser lines; each sample was assessed using a collection of 10,000 events. Each experiment was carried out in triplicate and the fluorescence was calculated using a FloMax^© Software.