Annexin 5 pe 7 aad apoptosis detection kit

Cell apoptosis was measured by the Annexin V-PE/7-AAD apoptosis detection kit (A213-01, Vazyme Biotech, Nanjing, China). MAC-T cells were harvested, and softly re-suspended in 1× binding buffer (100 μL). Then Annexin V-PE (5 μL) and 7-ADD (5 μL) were added to each group and incubated in the dark for 15 min. Approximately 10,000 or 20,000 cells from each group were used to analyze on a FACS Calibur system (BD Biosciences) and evaluated with the FlowJo software (version 10.0.7).

Kae (C₁₅H₁₀O₆; purity ≥97%), triphenyl-2,3,5-tetrazoliumchloride (TTC), methyl 3-{[(4-methylphenyl)sulfonyl]amino}benzoate (MSAB) (Wnt/β-catenin pathway inhibitor; purity ≥95%), D-hanks, trypsin, crystal violet, paraformaldehyde, and 4,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s Modified Eagle Media/Ham’s F-12 (DMEM/F12), B27, epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF) were purchased from Gibco (Grand Island, NY, USA). Glucose-free DMEM/F12 was purchased from Procell (Wuhan, China). Cell counting kit-8 (CCK-8) was obtained from MedChemExpress (Monmouth Junction, NJ, USA). Annexin V PE/7-AAD apoptosis detection kit, TUNEL BrightGreen apoptosis detection kit, bicinchoninic acid (BCA) protein quantification kit, and electro-chemi-luminescence (ECL) detection kit were acquired from Vazyme (Nanjing, China). Protein A/G agarose beads were obtained from Thermo Fisher Scientific (Waltham, MA, USA).

Flow cytometry analysis for cell apoptosis was performed using an Annexin V-PE/7-AAD Apoptosis Detection Kit (Vazyme, A213-01). In H₂O₂-induced apoptosis experiments, cells were treated with H₂O₂ with a final concentration of 400 μM for 4 h before apoptosis detection. Cells were digested with ethylenediaminetetraacetic acid (EDTA)-free trypsin (Macgene, CC035) for 3 min, collected by centrifugation, washed with ice-cold phosphate-buffered saline (PBS), and resuspended at a density of 5 × 10⁵ cells/ml with 100 µl 1× binding buffer. Then 5 μl Annexin V-PE and 5 μl 7-AAD were added and incubated for 10 min in the dark. Finally, cells were incubated with an additional 400 µl 1× binding buffer and analyzed within 20 min by CytoFLEX S (Beckman Coulter Life Science). At least 1 × 10⁴ cells were collected to determine the percentage of apoptotic cells. CytExpert (version 2.4) was used to analyze flow cytometry data. For TUNEL assays, the TUNEL Cell Apoptosis Detection Kit (KEYGEN, KGA703) was used according to the manufacturer’s protocol.

For cycle analysis, the test was performed using a cell cycle and apoptosis analysis kit (C1052; Beyotime, Shanghai, China). For apoptosis analysis, the test was performed using the Annexin-V-PE/7-AAD apoptosis detection kit (Vazyme Biotech, Nanjing, China). The transfected cells were harvested, washed, and stained according to the manufacturer’s protocol. Then, the stained cells were measured by CytoFLEX (Beckman Coulter, USA) and analyzed using FlowJo software version 7.6. Three independent assays were conducted.

Cell death was assessed using either the Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences, USA) or the Annexin V-PE/7-AAD Apoptosis Detection Kit (Vazyme, Nanjing, China) in accordance with the manufacturer’s instructions. The proportion of cell death was subsequently measured by flow cytometry. Cell viability was determined using the Cell Counting Kit-8 (CCK-8) (Dojindo, Japan) as per the manufacturer’s recommendation. To measure cell proliferation, the CCK-8 (Dojindo, Japan) was used following the manufacturer’s instructions.

Relevant cells were seeded in 6-well plates and treated as indicated. Annexin V/7-AAD staining was performed using Annexin V-PE/7-AAD Apoptosis Detection Kit (Vazyme, A213), following the manufacturer’s instructions.