Annexin 5 apc pi apoptosis detection kit

The chemicals and reagents are as follows: IMDM cell medium, F12 cell medium (Gibco); osteogenic/adipogenic/chondrogenic induced differentiation medium (Suzhou Syagen); II-type collagenase, LPS, 3-MA, IGF-1Triton, and X-100 Tween (Sigma); Rabbit type II collagen primary antibody, rabbit Cleaved Caspase-3 primary antibody, rabbit Bax primary antibody, rabbit LC3 primary antibody, rabbit Beclin-1 primary antibody, rabbit β-actin primary antibody, rabbit p-Akt primary antibody, rabbit Akt primary antibody, rabbit p-mTOR primary antibody, rabbit mTOR primary antibody, HRP-labeled goat anti-rabbit secondary antibody, rabbit CD45 primary antibody, rabbit CD34 primary antibody, rabbit CD29 primary antibody, rabbit CD90 primary antibody, rabbit CD63 primary antibody, rabbit CD9 primary antibody, Rabbit GAPDH primary antibody, and rabbit TSG101 primary antibody (Abcam); TBS buffer, PBS buffer (Biosharp); Exosome Isolation Kit (Life technologies); primary antibody dilutions, secondary antibody dilutions, Trizol Lysate, ECL kit, PKH67 Staining Kit, BCA Protein Quantitative Kit, Annexin V-APC/PI Apoptosis Detection Kit, DAPI staining solution, RIPA Lysate (Medium), and PMSF (Beyotime, Jiangsu); 5 All-In-One RT MasterMix, SYBR Green Supermix (Abm), PAGE Gel Rapid Preparation Kit (12.5%) (Shanghai Epizyme); TNF-α ELISA Detection Kit, IL-1β ELISA Detection Kit, and Shanghai Enzyme Link.

Cells in the exponential growth phase were digested with trypsin without EDTA and neutralized in complete medium. The single‐cell suspension was collected and centrifuged at 978 g for 5 min, and then washed with PBS for three times. The cells were stained with 500 μl binding buffer and gently mixed, after which 5 μl propidium iodide (PI) and 5 μl antigen‐presenting cell (APC)‐labelled Annexin V reagent (Annexin V‐APC/PI Apoptosis Detection Kit; Beyotime) was added and mixed thoroughly. The cells were then incubated for 15 min at room temperature. Apoptosis was detected by flow cytometry within 1 h. The above experiments were repeated three times.

To analyze apoptosis, transfected cells were harvested, and an Annexin V-APC/PI apoptosis detection kit (C1062M, Beyotime Co) was used as instructed in the manufacturer’s protocol. Then, apoptotic cells were analyzed with a FACS Calibur flow cytometer (BD Biosciences, New Jersey).
To analyze the intracellular and mitochondrial Ca²⁺ content, Fluo-2 (KGAF022, KeyGEN Co., Nanjing), and Rhod-2/AM (#40776ES50, Yeasen Co., Shanghai) were diluted to recommended concentration with 10% FBA culture medium and loaded into the transfected cells for 60 min at 25°C. Finally, the fluorescence intensity was detected by flow cytometry (BD Biosciences) and imaged with a Zeiss immunofluorescence microscope (Zeiss, Germany).
Total intracellular ROS levels were determined by staining cells with dichlorofluorescin diacetate (DCFH-DA, S0033, Beyotime). Briefly, cells were washed with PBS and incubated with 10 μM DCFH-DA at 37°C for 30 min. Cells were then washed twice with PBS and analyzed by flow cytometry (BD Biosciences).