Annexin 5 fitc pi

Annexin V-FITC/PI (Beyotime Biotechnology, Shanghai, China) was used to detect apoptosis activity using flow cytometry. Cells were treated according to the experimental design described previously. This was followed by two washes in PBS, digestion with trypsin, and resuspension in PBS. Then, 100 μL of the cell suspension was transferred to a 2-mL EP tube, and the cell density was increased to 1 × 10⁶ cells/mL. The supernatant was discarded after 5 min of centrifugation at 1000× g. The cells were gently resuspended in a mixture of 195 μL Annexin V-FITC conjugate, 5 μL Annexin V-FITC, 10 μL PI solution, and 400 μL PBS. After 20 min of incubation at 37 °C away from light, apoptosis was analyzed using the BD LSRFortessa flow cytometer (BD Company, Franklin Lake, NJ, USA).

Copper acetate was obtained from Macklin. Gallic acid (GA) was purchased from Kermel. Surfactant Aerosol OT was obtained from Alfa Aesar. Cisplatin was purchased from Shanghaiyuanye Bio-Technology Co., Ltd. l-arginine (l-Arg), NADH, glutathione (GSH) and H₂O₂ Assay Kit were obtained from Beijing Solarbio Science & Technology Co., Ltd. DSPE-PEG₂₀₀₀ was purchased from Top-Peptide Co., Ltd. BCA Kit, Lyso-Tracker Red, Hoechst 33342, DAPI, GSH Assay Kit, Calcein-AM/PI staining kit and Annexin V-FITC/PI were obtained from Beyotime Biotechnology. Charcoal-stripped fetal bovine serum (CS-FBS) was supplied by Hyclony. Nitric Oxide Assay Kit was obtained from Nanjing Jiancheng Bioengineering Institute. NO fluorescence probe (DAF-FM DA) was purchased from meilunbio. 4-hydroxyestradiol (4-OHE2) and 17β-estradiol (E2) were obtained from Cayman Chemical. PD98059 and Calpeptin were purchased from MCE. All other chemicals were provided by Sigma Aldrich (St. Louis, MO, USA) unless mentioned otherwise.

J774A.1 cells or PMs (5 × 10⁵ cells/well) were cultured in 12-well plates and treated with parasites for the indicated time periods. Cells were double-stained with Annexin V-FITC/PI (Beyotime, Shanghai, China) at room temperature (RT) in the dark for 20 min, and tested with flow cytometry using BD FACS Canto II (BD Biosciences, San Jose, CA, USA). The percentage of Annexin V-positive cells was quantified with BD FACSDiva software and results were processed by FlowJo software (Tree Star, Ashland, OR, USA).

AGS and HGC27 cells treated with berberine were harvested and cell apoptosis induced due to berberine was identified using the annexin V-FITC/PI (Beyotime, Shanghai, China) staining according to the instructions. The percentage of apoptotic cells was assessed using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA).

The apoptosis induction assay was operated using a BD Accuri C6 flow cytometry instrument and Annexin V-FITC/PI from Shanghai Beyotime Biotechnology Co., Ltd. (Shanghai, China) in China.
We also investigated whether S¹ could induce apoptosis using DMSO as a negative control. HepG2 cells (1 × 10⁶) were cultured in 35 mm dishes and incubated for 24 h at 37 °C. The cells were incubated with DMSO (5 μg/mL) and S¹ (12.5, 25, and 50 μg/mL) for 48 h (each concentration was repeated three times for optimal incubation time), washed, trypsinized (non-EDTA), and centrifuged (2000× g rpm/min). Cells were harvested and resuspended in 500 μL buffer containing Annexin V-FITC (5 μL of Annexin V-FITC and 5 μL of PI). The cells were stained with Annexin V-FITC and incubated for 5–15 min in the dark. Cells (1 × 10⁴) were collected for flow cytometry analysis with a single 488 nm argon laser.