Trypsin s310kj

Thirty-two male specific pathogen free (SPF)-grade BALB/C nude mice, approximately 6 weeks old, were obtained from Chongqing Ensiweier Biological Technology Co., Ltd., China. Mice were randomly divided into four groups (OE-NC, OE-CHMP4C, IN-NC, and IN-CHMP4C), each consisting of eight mice. The mice were acclimated in an animal facility at 25°C and a 12–12 h light-dark cycle for 1 week. MG63 cells overexpressing CHMP4C, MG63 cells with CHMP4C knockdown, and their corresponding control MG63 cells were digested with trypsin (S310KJ, Beyotime Institute of Biotechnology, China) to prepare cell suspensions at a cell density of 3×10⁷ cells/mL. The skin of the mice at the thigh root was disinfected and each mouse was subcutaneously injected with a cell suspension (0.5 mL). After 4 weeks, the mice were anesthetized and tumor images were observed and captured. Tumor dimensions were gauged, and euthanasia of the mice was carried out. The tumors were fully excised and weighed. Following that, RT-qPCR and immunofluorescence techniques were employed to assess the mRNA and protein expression levels. The animal study protocol was approved by the Ethics Committee of Affiliated Hospital of Qingdao University (QYFY WZLL 28188, 2023.11.06).

The flow cytometry was performed using Annexin V-APC/DAPI Apoptosis Kit as per manufacturer's instructions (E-CK-A258; Elabscience). After the experimental animals were anesthetized, the skull was opened to expose the brain, and the hippocampus was isolated and then put into in frozen PBS. The tissue was cut into small pieces of about 1 mm3, 0.25% trypsin (S310 KJ; Beyotime) was used for digestion at 37 °C for 20 min, and DMEM was provided to terminate the digestion. After being collected, the cell suspension was centrifuged at 1000 rpm for 5 min, added with PBS, and allowed to make a single cell suspension (4 × 10⁵ cells/mL), and we repeated centrifugation again at 300 g for 5 min, discarded the supernatant, resuspended cells and repeated again, then washed with PBS, and added 100 μL diluted 1×Annexin V Binding Buffer to resuspend cells in solution. 2.5 μL Annexin V-APC plus an equal quantity of DAPI staining solution was supplied, and the cell suspension was incubated in the dark at room temperature for 20 mi, added to 400 μL diluted 1×Annexin V Binding Buffer, and detected in a flow cytometer CytoFLEX (Beckman, USA).