Propidium iodide

Following 48 hours of transfection, the cells were harvested through centrifugation at 1000 rpm for 4 minutes. The cells were then fixed overnight at 4°C in 70% ethanol. After 24 hours of fixation, the cells were washed three times with PBS. Subsequently, the cells were stained with a solution containing 500 μl of propidium iodide (Elabscience, Wuhan, China) and RNase A at a ratio of 9:1. The cells were subjected to flow cytometric analysis using a flow cytometer (BD Biosciences, San Jose, CA, United States) following a 45-minute incubation at room temperature. After 72 hours of transfection, the cells were harvested by centrifugation (1000 rpm, 4 minutes) and washed thrice with PBS. Subsequently, the cells were resuspended in 500μl of binding buffer and double stained with Annexin V and PI, in accordance with the instructions provided by the FITC Annexin V/PI Apoptosis kit (Elabscience, Wuhan, China). Flow cytometry was then performed on the samples using the flow cytometer (BD Biosciences) after a 15-minute incubation in the dark at room temperature.

To confirm surface marker of MSCs, flow cytometry analysis was applied according to the manufacturer’s instructions. 1 × 10⁶ cells at P3 in the logarithm growth period were collected. After washing with 1% pre-cooled FBS/PBS and centrifuging at 350×g for 5 min, these cells were incubated with anti-CD45-APC (Invitrogen, USA), anti-CD29-FITC (Invitrogen, USA), and anti-CD44-APC (Novus Biologicals, USA) in the dark at 4 °C for 30 min, respectively. Labeled cells were washed twice and examined using the FACScan flow cytometry system (BD, Franklin Lakes, USA). FlowJo software (TreeStar, Ashland, OR, USA) was used to analyze the data. PBS solution was used as negative control. For cell cycle analysis of DNA content, the cells were cultured for 48 h under normoxia or hypoxia condition before they were collected, washed with PBS, and resuspended with 0.3 ml PBS and 1.2 ml pre-cooled 100 % ethanol for 1 h at − 20 °C. The cells were then centrifuged (300×g, 5 min) and resuspended with 1 ml PBS for 15 min at room temperature. The cells were then centrifuged (300×g, 5 min) again. One hundred microliters RNase A (Elabscience Biotechnology Co., Ltd., China) was added to each sample which was incubated at 37 °C for 30 min. Before test, 400 μl propidium iodide (Elabscience Biotechnology Co., Ltd., China) was added to each tube at 4 °C for 30 min.