Kga106

After transfection for 48 h, the cell cycle detection kit (KGA512, KeyGEN, China) and the apoptosis detection kit (KGA106, KeyGEN) were used to measure cell cycle distribution and apoptosis, respectively [22 (link)]. The cells used to detect cell cycle were washed with PBS and collected by centrifugation (310 g, 5 min). They were then fixed with 70% ethanol overnight at 4°C, rinsed, and suspended in 0.5 mL solution including propidium iodide (PI) and RNaseA (PI:RNaseA = 9:1). After incubation for 45 min at room temperature (RT), they were analyzed by FCM (C6, BD Sciences, USA).
The cells used to detect cell apoptosis were resuspended. Then, the Annexin V-FITC solution (5 μL) was added to cells and mixed, and then PI (5 μL) was added. After reaction at RT for 10 min in the dark, the apoptosis rate was assessed via FCM.

Cell apoptosis was evaluated via apoptosis kit (KGA106, KeyGen). A875 and SK-MEL-1 cells (5 × 10⁵) in 200 µL binding buffer reacted with 10 µL annexin V-fluorescein isothiocyanate (annexin V-FITC) and 10 µL propidium iodide (PI). After co-incubation for 20 min, cell status was detected through the flow cytometer (BD Biosciences, San Diego, CA, USA) and apoptosis rate was expressed as the ratio of apoptotic cells in total cells.
Cell cycle was determined by Cell Cycle Detection Kit (KGA511, KeyGen). A875 and SK-MEL-1 cells (1 × 10⁶) were centrifuged at 2000 rpm for 5 min and cell pellets were fastened with 500 µL 70% cold ethanol (Sigma) at 4 °C overnight. Then, cells were washed with 200 µL phosphate buffer solution (Gibco) and added with 500 µL PI/RNase A working solution for 45 min. The red fluorescence at excitation wavelength 488 nm was examined under the flow cytometer (BD Biosciences), and DNA content was analyzed in different phases (G1, S, G2, M).