Apoptosis and Mitochondrial Membrane Potential Analysis

Flow cytometric analysis of apoptosis was performed using the fluorescein isothiocyanate (FITC) Annexin V Apoptosis Detection kit I (BD Biosciences, San Jose, CA, USA) and performed as previously described (27 (link)). Briefly, cells were dissociated with trypsin and resuspended at 1×10⁶ cells/ml in binding buffer with 50 µl/ml FITC Annexin V and 50 µl/ml propidium iodide (PI). Cells were subsequently incubated for 15 min at room temperature and analyzed using a Gallios flow cytometer (Beckman Coulter, Inc., Brea, CA, USA). The cell's inner mitochondrial membrane potential (Δψm) was detected by flow cytometry using a MitoScreen JC-1 staining kit (BD Biosciences) following a previously described protocol (27 (link)). Briefly, cells were dissociated with trypsin, resuspended at 1×10⁶ cells/ml in assay buffer and then incubated at 37°C for 15 min with 10 µl/ml JC-1. Cells were washed twice with the assay buffer prior to analysis with the flow cytometer. Flow cytometric data were analyzed using FlowJo 7.6 software (FlowJo LLC, Ashland, OR, USA).