Annexin 5 fitc and propidium iodide kit

Cell apoptosis was evaluated using Annexin V-FITC and Propidium Iodide (PI) Kit (Invitrogen) by flow cytometry as previously described [17 (link)]. After being washed with PBS and resuspended with binding buffer, a mixture containing 5 μL of Annexin V-FITC and 5 μL of PI was added to the 1000 μL of cell resuspension with 1 × 10⁶ cells (in Annexin-V binding buffer) and incubated for 30 min in the dark. The stained cells were centrifuged and washed twice with Annexin-V binding buffer and resuspended in a 400 μL of Annexin-V binding buffer. Finally, the percentage of apoptotic cells was detected by a flow cytometer (BD Biosciences).

MN9D cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA), penicillin (100 IU/ml), and streptomycin (100 mg/ml) at 37°C in a humidified incubator with 5% CO₂. For cell treatment, MN9D cells were treated with MPP⁺ (Sigma–Aldrich, St. Louis, MO, USA) at different concentrations for indicated times. Bafilomycin A1 (100 nM, Selleck Chemicals, Shanghai, China) was used in this study. Cell death assays were analyzed by flow cytometry (Becton-Dickinson Immunocytometry Systems, San Jose, CA, USA) using an Annexin V-FITC and propidium iodide (PI) kit (Invitrogen, Carlsbad, CA, USA).