T2600000

The apoptosis of PC-12 cells was measured by Annexin V-FITC/PI apoptosis detection kit (40302ES20, Yeasen, Shanghai, China). After transfection and LPS treatment, PC-12 cells were digested in EDTA-free trypsin (T2600000, Sigma-Aldrich, USA), and washed with phosphate buffered saline (PBS; AM9624, Thermo Fisher, USA) thrice. The cells were then resuspended by 1× Binding Buffer to be 1 × 10⁶ cells/mL and added with Annexin V-FITC solution (5 μL) and propidium iodide (PI) solution (10 μL). After 10 min incubation in the dark, apoptotic cells were examined by a flow cytometer (CytoFLEX, Beckman Coulter, Brea, CA, USA).

A TUNEL assay kit (ab66108, Abcam, Cambridge, MA, USA) was used to assess cell apoptosis in rat cartilaginous tissues. Rat cartilages were chopped into 0.5 mm thick tissues, then washed with PBS containing 10 mL/L penicillin-streptomycin (P4333, Sigma-Aldrich, USA), and digested in 0.25% trypsin (T2600000, Sigma-Aldrich, USA) for 1 h. Subsequently, digestion with type II collagenase was performed at 37°C overnight. When single cells were captured under an inverted microscope (IX71; Olympus, Tokyo, Japan), Dulbecco's modified Eagle medium (DMEM; A4192101, ThermoFisher, USA) was added to stop the digestion, and chondrocytes were harvested after centrifugation at 1000 × g for 5 min. Following fixation with 4% paraformaldehyde for 15 min on ice, chondrocytes were washed with PBS, incubated with ice-cold 70% ethanol for 30 min, and separated by 0.1% Triton X-100 (X100, Sigma-Aldrich, USA). Then, apoptotic chondrocytes were colored in staining solution at 37°C for 60 min and subsequently counterstained by 4′,6-diamidino-2-phenylindole (DAPI, D9542, Sigma-Aldrich, USA) for 7 min. The fluorescent chondrocytes were captured by an inverted fluorescence microscope (ECLIPSE Ti, Nikon, Tokyo, Japan) under ×200 magnification.