Cell apoptosis detection kit

Intestinal macrophages were inoculated into 6‐well plates and stimulated with LPS for 12 h. Both suspended and adherent cells were collected, washed with PBS for two times, resuspended in pre‐cooled PBS. After centrifugation, cells were suspended in binding buffer and subjected to the cell apoptosis detection kit (BD, Massachusetts, USA) was used for detection. 5 μl of Annexin V‐FITC and 5 μl of PI staining were added to the cell suspension and subjected to flow cytometry after incubation in dark for 15 min.

To be specific, KCs were inoculated into the 6‐well plates and grown to 80% before drug intervention. At 12 h after LPS induction, all cells were collected, which were washed with the pre‐chilled PBS, and centrifuged at 1500 g for 30 min. Thereafter, cells were stained after suspension with Binding Buffer, and incubated with 5 μl Annexin V‐FITC for 5 min in dark and then with 5 μl PI for another 5 min in dark in cell apoptosis detection kit (BD, Massachusetts, USA). After PBS washing, the cells were loaded for detection using the Annexin V‐FITC (+) PI (+) and Annexin V‐FITC (+) PI (−) apoptosis cytometers.

Cell apoptosis detection kit (propidium iodide (PI), RNase staining buffer and FITC-labeled Annexin V) were purchased from BD Pharmingen (San Diego, CA, USA). Cells were seeded 2.5 × 10⁵ per well in 6-well plates for 24 h. Then the medium was replaced by culture medium containing metformin 0, 20 or 40 mM for 24 or 48 h. The cells were harvested for analysis of cell cycle and apoptosis, respectively. The cell cycle was analyzed using PI staining, according to the manufacturer’s instructions. Briefly the cells were fixed in 70 % ethanol, stained with PI, and the amount of PI-labeled DNA in a cell was measured by a flow cytometer (Accuri C6, Becton Dickinson, San Jose, CA, USA). The acquired data were analyzed by FlowJo software (Ashland, OR, USA). To determine the apoptotic cells, the cells were stained with Annexin V-FITC and PI immediately after harvesting, and analyzed by flow cytometry, as described by the manufacturer’s instructions.

The apoptosis rates of PC9 and Calu-3 cells were assessed using the Cell Apoptosis Detection Kit (BD, USA). Cells (6 × 10⁴) were harvested at logarithmic growth phase and suspended in 100 µL of the binding buffer. After that, the cells were incubated with a mixture containing 5 µL/well Annexin V-fluorescein isothiocyanate (FITC) and 5 µL/well propidium iodide (PI) in the absence of light for 15 min at 25°C. After incubation, the cells were washed three times with PBS and suspended in 400 µL of the binding buffer. Flow cytometry was used to detect the cells. The cell apoptosis rate was analyzed by summing the percentage of upper-right and lower-right quadrants.