A211 01

The cells were cultured in 12-well plates, digested with trypsin without EDTA, and collected after digestion. Centrifugation was performed at 1000 rpm at 4°C for 5 min, and the supernatant was discarded. The cells were washed twice with precold PBS at 1000 rpm and centrifuged at 4°C for 5 min, and the supernatant was discarded. The cells were suspended by 100 μM 1x binding buffer. Annexin V-FITC 5 μM and PI staining solution 5 μM were added and incubated at room temperature for 10 min (Vazyme, a211-01), protected from light. Add 400 μM 1x binding buffer and mix. C6 flow cytometry was used for detection, and FlowJo (version 10) was used for analysis.

Cell was collected by trypsin digestion and washed with PBS twice. The cell was stained according to the manufacturer’s protocol (Vazyme, Nanjing, China, A211-01) and analyzed by flow cytometry (Accuri, MI, United States).

LX-2 and L-O2 cells in the logarithmic growth phase were collected, and cells were counted and seeded into six-well plates at 3 × 10⁵ cells per well. After cell attachment, the medium in each well was replaced with DMEM complete medium containing different concentrations of SEA (0, 5, 10, or 20 μg/mL), and subsequent flow cytometric assays were performed after 48 h of stimulation (Nanjing Vazyme Biotechnology, A211-01, Nanjing, China). Cells in 6-well plates were collected in flow tubes; cells were washed twice with precooled PBS; and supernatants were removed by centrifugation at 4 °C. Subsequently, 100 μL of 1× binding buffer was added, and cells were gently pipetted to form a single cell suspension. Then 5 μL Annexin V-FITC and 5 μL PI staining solution were added to each experimental-group tube, whereas untreated cell samples were used as a negative control. The experimental group tubes were stimulated with 20 μg/mL SEA for Annexin V-FITC and PI single staining for compensation adjustment, while gently blowing on the well; cells were incubated at room temperature in the dark for 10 min, and 400 μL 1× binding buffer was added and gently mixed. The stained samples were then detected by flow cytometry within 1 h.