Annexin 5 pi

Cell apoptosis was analyzed by the Annexin V-PI (Beyotime Institute of Biotechnology) staining kit according to manufacturer's instructions. Briefly, after treatment in diverse groups, HUFs were harvested and washed three times with cold PBS, and then were resuspended in 500 μL binding buffer. Afterward, each cell sample (200 μL cell suspension) was added with 5 μL Annexin V-FITC followed by 10 μL propidium iodide for 10 min incubation in the dark at 25 °C for 15 min. Finally, cell apoptosis was performed on a FACS flow cytometer (Becton Dickinson) and were analyzed with FlowJo software (Tree star, Ashland, Oregon).

KYSE30 and KYSE150 cells were seeded at a density to achieve 70% confluence into 12-well plates. After cell adhesion, cells were treated with different concentrations of drugs for 12 h. To measure cell death, Annexin V/PI (C1062, Beyotime) was used for the detection of cell apoptosis or necroptosis. In addition, the cells were stained with 5 μg/ml propidium iodide (PI), and the percentage of the PI-positive cells was analyzed using a BD Accuri C6 flow cytometer. There were 1 × 10⁴ cells counted per sample. PI-positive cells may have undergone apoptosis, pyroptosis, necroptosis, or ferroptosis. For live cell imaging, 100 nM SYTOX Green (S7020, Thermo Fisher Scientific) was added for 20 min, then photographed using a Lionheart FX (BioTek). The resulting images were analyzed by ImageJ software, and the percentage of green-positive cells in each image was calculated.

Apoptotic cells were quantified by surface annexin V-PI staining. AN3CA cells (2×10⁵ cells/group) after 96 h transfection and untransfected cells with or without 48 h sitagliptin treatment were collected using 0.05% trypsin, with the supernatant used to terminate the digestion. Cells were washed with PBS and resuspended in binding buffer composed of 10 mmol/l HEPES (pH 7.4), 2.5 mmol/l CaCl₂ and 140 mmol/l NaCl. 195 μl Apoptosis Assays Buffer was added to cells, followed by incubation with 5 μl annexin V-PI (C1065; Beyotime Institute of Biotechnology, Shanghai, China) for 15 min in the dark at room temperature. A total of 10,000 cells were acquired per sample and data were analyzed using Cell Quest software (BD Pharmingen, San Jose, CA, USA).

Annexin V/PI and Annexin V/7-AAD Staining Kit (Beyotime) were used to detect cell apoptosis. Splenic cells or peripheral blood in mice were stained with anti-CD3 (APC), anti-CD4 (PE-CY7) and anti-CD8 (BV510) antibodies (Biolegend), as well as Annexin V-PE and 7-AAD. Human peripheral blood was stained with anti-human CD3 (FITC), anti-human CD4 (PE-CY7) and anti-human CD8 (BV510) antibodies as well as Annexin V-PE and 7-AAD. Purified CD3⁺ T cells obtained from MRL/lpr mice were incubated with DMSO or MLN4924 (0.1 and 0.5 μM) for 12 h and then stained with the indicated antibodies: anti-CD3 (APC), anti-CD4 (PE-CY7) and anti-CD8 (BV510) (Biolegend). Peripheral Blood Mononuclear Cell (PBMC) isolated from patients were treated with 0.5 μM MLN4924 for 6 h and then stained with anti-human CD3 (APC-CY7), anti-human CD4 (PE-CY7) and anti-human CD8 (BV510) antibodies as well as Annexin V-FITC and PI-PE to detect cell apoptosis. All these cells were analyzed with Beckman CytoFlex S system (Beckman).

The Annexin V/PI assay was conducted according to the manufacturer's instructions (Beyotime). Briefly, cells were transfected with WIF expressing plasmid and then treated with gossypol for 24 hours at 37°C. Afterwards, cells were washed with prechilled PBS, trypsinized for 1 min, and resuspended in 100μl of binding buffer supplemented with 2.5μl FITC conjugated Annexin-v and 1μl PI (100 μg/ml). Then, cells were shaken at room temperature for 15 min in darkness. A total of 10, 000 cells were collected and assessed by flow cytometry (BD Biosciences).

After collection, cells were stained with Annexin V-PI (Beyotime Institute of Biotechnology) based on instructions and then sorted by flow cytometry (FACScan, BD Bioscience, N.J., USA). Results were evaluated by CELL Quest 3.0 software (BD Bioscience, N.J., USA). When Annexin V was used in combination with PI, PI was excluded from viable cells (FITC-/PI-) and early apoptotic cells (FITC + /PI-), while late apoptotic cells and necrotic cells were both positive for FITC and PI.

The change in nucleus and the fluorescence intensity were observed by means of DAPI, JC-1 and Annexin V/PI (Beyotime Institute of Biotechnology, Shanghai, China) staining assay after resistomycin treatment for 24 h, which we conducted as previously described [18 (link)].