4.4.2. Evaluation of Cell Death Status

Cells were washed with 200 µL of cold Annexin V binding buffer twice, after the splitting step (described in cell cycle analysis) and incubated with 50 µL of Annexin V binding buffer containing 5 µL of PI and 2.5 µL of APC AV at 37 °C for 15 min. Then, 450 µL of AV binding buffer was used to resuspend cells prior to the analysis. Lastly, the cells were subjected to flow cytometry analysis by evaluating cell population in both FL3-H (a 488 nm laser for excitation and a long pass filter at 670 nm for emission) and FL-4-H channel (a 633 nm laser for excitation and a bandpass filter at 661/16 nm for emission).