Macs flow cytometer

Quantitative changes in ΔΨm at the early stage of cell apoptosis were measured using a J aggregate-forming lipophilic cation-1 (JC-1) Mitochondrial Membrane Potential Detection kit (Nanjing KeyGen Biotech Co., Ltd.) according to the manufacturer's instructions. Cells were treated with 4, 8, or 12 µM LFG-500 for 24 h, then digested with trypsin (Sigma-Aldrich; Merck Millipore) and centrifuged at 367 × g for 5 min at 4°C. The harvested cells were washed once with PBS and incubated with 10 µM JC-1 dye in incubation buffer at 37°C for 20 min in the dark. Subsequently, the cells were washed twice with pre-chilled buffer solution. Relative fluorescence intensities of the cells were monitored using a MACS Flow Cytometer (Miltenyi Biotec GmbH) with MACSQuantify Software (Miltenyi Biotec GmbH; software version 2.4).

Following treatment with LFG-500 (4, 8 or 12 µM) for 24 h, cells were harvested and washed with PBS. As previously described (21 (link)), apoptotic cells were identified by double supravital staining with the Annexin V-FITC apoptosis detection kit according to the manufacturer's protocol. Briefly, the cells were resuspended in 500 µl binding buffer, and 5 µl Annexin V-FITC staining solution was added and gently mixed. Subsequently, 5 µl propidium iodide (PI) staining solution was added and mixed evenly with the cells. Finally, the cells were incubated in the dark for 15 min at room temperature, and after 1 h, the rate of cell apoptosis was evaluated and analyzed using a MACS Flow Cytometer with MACSQuantify Software (version 2.4) (both from Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Early apoptotic cells were Annexin V-positive and PI-negative, whereas late apoptotic cells were Annexin V and PI-positive.