Fitc annexin 5 apoptosis detection kit 1

Apoptosis was detected with flow cytometry analysis. Briefly, cells were collected by trypsinization at 48 h after treatment with IL-6, osimertinib, or both. Then, cells were washed three times with PBS, and resuspended at a density of 1 × 10⁶ cells/mL. Next, cells were double-stained with PI for 10 min at ambient temperature in the dark using FITC Annexin V Apoptosis Detection Kit I (Sigma, Germany). Then, cells were analyzed using a flow cytometer (Beckman Coulter Navios, CA, USA).

For apoptosis, the cells (1 $\times {10}^6$ cells) were suspended with 500 $\mu L$ binding buffer and with 5 $\mu L$ of Annexin V-FITC and 10 $\mu L$ of PI (FITC Annexin V Apoptosis Detection Kit I, Sigma Aldrich, St. Louis, USA) added, and were later mixed. Next, the suspended cells were incubated at 4 ${}^{\circ }$ C for 30 min in dark. For the cell cycle, the cells were suspended with 500 $\mu L$ of binding buffer and assayed by PI/RNase Staining Buffer (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s instruction. The apoptotic cells were analyzed using EXPO32 ADC, an analysis software (Beckman Coulter, Brea, CA, USA). The cell cycle was analyzed using ModFit 3.0 analysis software (Verity Software House, Inc. Topsham, ME, USA).