Lx flow cytometer

The mitochondrial membrane potential (MtMP) was measured by the fluorescence of the potential-dependent TMRE (tetramethylrhodamine ethyl ester). Briefly, cells were incubated with 500 nM TMRE at 37 °C for 30 minutes, protected from light. After staining with TMRE, fluorescence was measured using flow cytometry. Cells were collected, suspended in phosphate-buffered saline (PBS), and analyzed with a CytoFLEX LX flow cytometer. Data analysis was performed using CytExpert software.

Cell apoptosis was measured using the Annexin V-FITC apoptosis detection kit. In practice, A549 cells were seeded into 6-well plates at density 1×10 5 cells per mL and were incubated for 4 h at 37 °C. Afterwards, the cells were treated with varying peptides doses for 4 h. After peptide incubation, the cells were washed twice with cold PBS and were collected by centrifugation. The cell suspensions were incubated with binding buffer (300 µL) and Annexin V-FITC (10 µL) for 20 min as well as PI (1 µL) for 10 min at room temperature in the dark. The fluorescence of the peptide-treated cells was measured by the CytoFLEX LX Flow cytometer. The data were analyzed with the CytExpert software. The FITC-conjugated Annexin V and the PI dye were excited at wavelength 488 nm. The emissions were recorded at wavelengths 525 nm and 585 nm for the FITC-conjugated Annexin V and PI, respectively.