Annexin 5 pi kit

HK‐2 cells were seeded at a density of 4.0 × 10⁵ per well. After the treatment of LPS, HK‐2 cells were harvested and suspended in binding buffer. Then, Annexin V/PI kits (KeyGEN, Nanjing, China) were applied for the detection of apoptosis. Flow cytometric analysis for the apoptotic rate were conducted using a FACSAria flow cytometer (BD Biosciences, San Jose, CA, USA). To detect the apoptosis levels of renal tissues, we applied the TUNEL assay. To quantify the apoptosis level of tissues, we analyse the brown TUNEL positive cells per field in each group.

The proliferation of ESCC cells was detected by the Cell Counting Kit-8 (CCK-8, Beyotime Institute of Biotechnology, Jiangsu, China). Briefly, cells were placed into 96-well plates at the density of 1000 cells per well before addition of CCK-8 and the absorbance was measured at a wavelength of 450 nm. Five replicates were conducted for cells in each group and the experiments were repeated three times. For the clone formation assay, cells transfected with the indicated lentivirus were seeded in six-well plates in triplicates at a density of 500 cell per well and incubated at 37 °C with 5% FBS for 14 days. The cells were fixed with 4% polyoxymethylene and stained with 1.5% methylene blue. The colonies were counted and analyzed by Image J software (NIH, Bethesda, MD, USA). For cell cycle analysis, the indicated cells were collected, rinsed with cold PBS twice, and labeled with propidium iodide (PI) solution (BD Cycletest Plus DNA Kit, San Jose, CA, USA). For cell apoptosis analysis, Annexin V/PI kits (KeyGEN, Nanjing, China) were used to detect apoptotic cells. The cells were then analyzed by a fluorescence-activated cell sorter according to a previous report.^{21 (link)}

D-pinitol (purity: 95%, Lot No: 441252) and streptozotocin (STZ) were from Sigma Chemicals Co. (St. Louis, MO, USA). Paraformaldehyde (PFA), dimethyl sulfoxide (DMSO), bovine serum albumin (BSA), trypsin/EDTA solution, D-glucose, and 3-(4,5-dimethylthiazol)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) were purchased from Solarbio (Beijing, China). Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were obtained from GIBCO (Grand Island, NE, USA). Hoechst 33258 and PI were purchased from Beyotime (Shanghai, China). AGE-BSA was purchased from Abcam (Cambridge, UK). ELISA kits and necrosulfonamide (NSA) were purchased from R&D systems (Minneapolis, MN, USA). Annexin V/PI Kit was from Key Gen (Nanjing, China). The primers were synthesized by Sangon Biotechnology (Shanghai, China). The MFG-E8 antibody was purchased from MBL (Woburn, MA, USA) and R&D (Minneapolis, MN, USA), and antibodies RIPK1, MLKL, phospho-MLKL, and β-action were purchased from Abcam (Cambridge, UK). RIPK3 and HMGB1 antibodies were purchased from Proteintech (Wuhan, China). All other reagents are standard commercial high purity materials.