Annexin 5 fitc pi apoptotic detection kit

Aβ_25-35 (Lot: Y-0044) was purchased from BIOSS Biotech Limited Company (Beijing, China). The Hoechst 33342/PI apoptotic assay kit was purchased from Sigma-Aldrich (St. Louis, MO, USA). The TUNEL apoptosis detection kit was purchased from BOSTER Biotech Limited Company (Wuhan, Hubei, China). The Annexin V-FITC/PI apoptotic detection kit was purchased from KeyGEN Biotech Corp., Ltd. (Nanjing, China). Rhodamine 123 was purchased from Yansheng biochemical reagent Co., Ltd. (Shanghai, China). The RT-PCR kit was purchased from TransGen Biotech Limited Company (Beijing, China). 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) was purchased from Sigma-Aldrich (Milan, Italy). Anti-NF-κBp65 antibody was purchased from Invitrogen (Carlsbad, CA, USA). The CO₂ incubator and ultra-low temperature freezer were purchased from SANYO Electric Co., Ltd. (Moriguchi, Japan). The laser scanning confocal microscope was purchased from Leica DMIRB (Rueil Malmaison, France). The flow cytometer was purchased from Becton-Dickinson Labware (Franklin Lakes, NJ, USA). The gel imaging and analysis system ChemiDocXRS were purchased from Bio-Rad Laboratories, Inc. (Berkeley, CA, USA). The TC-512PCR amplification instrument was purchased from TECHNE (London, UK).

Apoptosis was assessed using an Annexin V‐FITC/PI apoptotic detection kit (Keygen, Biotech), according to the manufacturer's instructions. Briefly, PC‐9 cells, HCC827 cells, PC‐9‐derived LCSCs or HCC827‐derived LCSCs, administered anlotinib (5, 10 or 20 μM) for 3 days, were incubated with FITC‐conjugated Annexin V and PI at room temperature for 15 minutes away from light. Then, these cells were subjected to flow cytometry (BD FACSCalibur; BD Bioscience) and sorted into intact (Annexin V and PI double‐negative), early apoptotic (Annexin V‐positive), late apoptotic (Annexin V and PI double‐positive) and necrotic (PI‐positive) cells.