Apodetect kit

Cells were seeded at 5,000 to 10,000 cells per cm² under standard conditions 24 h prior to treatment with 100 nM of anti-ErbB2 agents. After 3 days, cells were collected by trypsinization and fixed in 70% EtOH or in 4% paraformaldehyde for propidium iodide (20 μg ml⁻¹; 2 Kunitz units per ml of RNAse) or TUNEL labelling (In situ cell death detection kit, Roche), respectively. The AnnexinV/PI assay was performed with non-fixed cells treated for 2 days (ApoDETECT Kit; Life Technologies). The cell cycle distribution and apoptosis rate were quantified with FlowJo software.

The cell surface exposure of phosphatidylserine on the plasma membrane of cells were assessed using Annexin V-FITC Apoptosis Detection Kit (Life-Technologies Apo-Detect Kit). Briefly, cells were harvested on a coverslip sited in a 24-well plate at a density of 10⁴ cells per well. After adhesion, cells were treated with phenformin at 0, 1 and 10 mM. After 24 hours of treatment cells were washed with PBS and supernatants were conserved for cyto-spin. Coverslips were incubated with a mix of Annexin V-FITC, PI and Hoechst 33258 (Thermofisher) in the dark for 10 min at room temperature. The same procedure was reserved for cells recovered in the supernatants after centrifugation. Cells were fixed with PFA 4% for 10 minutes. After washing with PBS, coverslips were mounted with Dako and the fluorescence images were obtained with a Leica TCS-SP5 II inverted confocal microscope.