Apoptosis and ROS Analysis in HaCaT Cells

Annexin V-PI Apoptosis Detection Kit (Sangene, Tianjin, China) and a ROS assay kit (EMD Millipore, Billerica, MA, USA) were utilized to perform apoptosis analysis as per the protocols provided by the manufacturer [45 (link), 46 (link)]. Concisely, HaCaT cells were plated into 6-well or 96-well tissue culture plates (triplicate) at a concentration of 5 × 10⁴cells/cm^2, followed by culturing in DMEM comprising 10 percent FBS for 24 hours. After cultivation, aspiration of the culture medium was carried out, and the cells were rinsed in PBS and subjected to culturing in DMEM comprising 2 percent FBS in the presence or absence of (control) 30 mM glucose for an extra 12-72 h. At the latest stage of cultivation, propidium iodine-Annexin V staining (San Jian, Tianjin, China) and reactive oxygen species (ROS) staining (Millipore) were conducted to determine apoptosis and ROS generation, respectively, at the specified time points following the manufacturer’s guidelines. Flow cytometry (BD Biosciences) was subsequently used to analyze the cells. For the detection of ROS, the probe dilution was done to a final concentration of 10 μM using a serum-free medium. The probe was introduced to the cells, placed in an incubator at 37° C with 5% CO₂ for 30 minutes, and then washed with a serum-free medium. Flow cytometry (BD Biosciences) was then employed to examine the cells.