Annexin 5 propidium iodide detection kit

Cell cycle was evaluated using the RNase A/Propidium Iodide Detection kit (KeyGEN, China). The staining buffer of PI/RNase A was prepared at the proportion was 9:1. After precipitated by centrifugation, the cell pellets were suspended using 500 μl of 70% cold ethanol, and then fixed for 2 h to overnight at 4 °C. After washing with PBS before staining, 500 μl of precooled PI/RNase A staining solution was added, mixed, and reacted at room temperature to avoid light for 30–60 min. The percentage of G0/G1, S, and G2/M phases were estimated using flow cytometry (BD LSRFortessa, USA). Cell death was determined using the Annexin-V/Propidium Iodide Detection kit (KeyGEN, China). The detached cells in the culture medium were collected first, and the attached cells were collected after trypsin digestion. The cells were precipitated using centrifugation and resuspended with 500 μl Binding Buffer. 5 μl of Annexin V-FITC and 5 μl of propidium iodide (PI) was added and mixed gently. The reaction was conducted in the dark at room temperature for 5–15 min, and the percentage of early, late apoptosis, viable and dead cells was detected by flow cytometry (BD LSRFortessa, USA) within 1 h.

Cells were plated at a density of 5×10⁵/cm² in a six-well plate, and incubated at 37 °C in a CO2 incubator for 12 h, allowing cells to completely adhere and spread on the six-well plate. The cells grew and were maintained in serum-free DMEM for more than 24 h to create a confluent monolayer. The confluent monolayer was scraped with a sterile toothpick and washed with PBS. Serum-free DMEM was then added and the width of the wound gaps were measured using NIH Image J analysis and normalized to the time 0 wounds for four independent experiments. Apoptosis after transfection treatment was examined by using the Annexin V/Propidium Iodide Detection Kit (KeyGEN) according to the manufacturer's instructions.