Cellquest version 5

The quantitative assessment of apoptotic cells was assessed using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) method, which enabled examination of DNA-strand breaks during apoptosis using a BD ApoAlertTM DNA Fragmentation Assay kit (BD Biosciences, Franklin Lakes, NJ, USA). The cells were trypsinized, fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton-X-100 in 0.1% sodium citrate. Subsequent to being washed with PBS three times, the cells (2×10⁵ cells/well) were incubated with the reaction mixture for 60 min at 37°C. The cells were immediately analyzed using a FACScan flow cytometer and the CellQuest version 5.1 (BD Biosciences).

To detect cell apoptosis, INS-1 cells were transfected with TRIAP1-siRNA, control-siRNA, TRIAP1-plasmid and control-plasmid, and then cultured for 48 h. Following transfection, the Annexin V/PI Cell Apoptosis Detection kit (Nanjing KeyGen Biotech Co., Ltd) was used to determine the percentage of apoptotic cells. A total of 1x10⁶ INS-1 cells were harvested and resuspended in 500 μl binding buffer containing 5 µl Annexin V and 5 µl propidium iodide. The resulting solution was incubated for 15 min in the dark and the BD FACSCalibur™ flow cytometer (BD Biosciences) was used to evaluate the fluorescence intensity. Data were analyzed using the CellQuest™ version 5.1 software (BD Biosciences).

Cell apoptosis was assessed using the Annexin-V/PI Apoptosis Detection kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. Briefly, cells were seeded (2×10⁵ cells/well) into 6-well plates and cultured at 37°C overnight. Following transfection, cells were harvested, centrifuged at 1,000 × g at 4°C for 5 min), and the cell pellet was resuspended in 100 µl FITC-binding buffer. Subsequently, the cell suspension was incubated with 5 µl ready-to-use Annexin V-FITC (BD Bioscience) and 5 µl PI at room temperature in the dark for 30 min. Cell apoptosis (late or early + late apoptosis) was assessed via flow cytometry using a BD FACSCalibur flow cytometer (BD Biosciences). Data were analyzed using CellQuest™ version 5.1 software (BD Biosciences).

Flow cytometry analyses were performed as described previously (25 (link)). In short, following the different treatments, PC12 cells were harvested and re-suspended in a 500 µl binding buffer. The cells were labeled with 5 µl Annexin V-FITC (Nanjing KeyGen Biotech. Co. Ltd.) and 5 µl of propidium iodide for 10 min in the dark. The apoptotic index was determined using a flow cytometer (BD Biosciences). All data were acquired and analyzed with Cellquest version 5.1 software (BD Biosciences).

Cells (KCs or T cells) were digested for 3 min in 0.25% trypsin at 37°C and collected following centrifugation at 50 × g for 5 min at room temperature. Cells were then rinsed in pre-cooled PBS (4°C) twice. Cells were then resuspended in 400 μl binding buffer and the cell concentration was diluted to 1×10⁶/ml. Fluorochrome-labeled mAbs, including CD14-phycoerythrin (PE; cat. no. Sa2-8; 12-0141, dilution, 1:100), CD163-APC (cat. no. GHI/61; 17-1639-42, dilution, 1:100), Foxp3-PE (cat. no. 150D/E4; 12-477, dilution, 1:100), CD25-fluorescein isothiocyanate (FITC; cat. no. CD25-4E3; 11-0257, dilution, 1:100), were purchased from eBioscience; Thermo Fisher Scientific, Inc. KCs were stained for CD14 and CD163 and T cells were stained for CD25 and Foxp3 for 1 h at 4°C in the dark. Flow cytometric cell sorting was performed using FACSAria; flow cytometric data were acquired using a FACSCalibur and analyzed using Cellquest version 5.1 software (BD Biosciences, Franklin Lakes, NJ, USA). Experiments were performed in triplicate.