4 6 diamidino 2 phenylindol dapi

Fluorescence intensity was determined using 2,7-dichlorodihydrofluorescein diacetate (H2DCF-DA; Invitrogen). To measure CRH-induced ROS formation, DPCs and ORSCs were seeded in 6-well plates at a density of 5 × 10⁴ cells/well. The DPCs and ORSCs were treated with 0.2–0.5 μM and 0.1–0.2 μM CRH, respectively, for 48 h at 37°C in a humidified atmosphere with 5% CO₂. To assess the effects of ROS scavenging by the GL extract, DPCs and ORSCs were initially treated with 0.5 and 0.2 μM CRH, respectively, for 24 h. Subsequently, the cells were incubated with CRH in the presence or absence of the GL extract (20 μg/ml) for an additional 24 h. Cells were treated with H₂O₂ (200 μM) as a positive control for 2 h at 37°C. After treatment, the cells were washed twice with PBS and incubated with 20 μM DCF-DA in PBS for 30 min at 37°C in a humidified atmosphere with 5% CO₂. After incubation, the cells were washed twice with PBS. Nuclei were counterstained with 4′,6-diamidino-2-phenylindol (DAPI, 1:1000, Invitrogen) for 1 min. The cells were washed again with PBS, and cytoplasm DCF-DA and nuclear DAPI staining was imaged using an inverted fluorescence microscope (Leica).

The BMSCs were implanted on plates and held in full culture medium for 1, 3, and 7 days. The cytoskeleton morphology was evaluated, where the cells were washed with cold 1X PBS for 2–3 min, fixed in 4% paraformaldehyde (PFA) at room temperature (RT) for 15 min and permeabilized with phosphate buffered saline containing 0.1% Triton X (PBS-Tx-0.1%). The cytoskeleton was stained with tetramethylrhodamine isothiocyanate (TRITC)-phalloidin (Yeasen, China), while the nuclei were stained with 4′,6-Diamidino-2-phenylindol (DAPI, ThermoFisher Scientific, USA). The samples were imaged using a fluorescence microscope (Olympus, Japan). The BMSC proliferation induced by different pore channels was evaluated, where cells were cultured in EdU-containing medium (diluted to 1: 1000, 1 mg L-1; Ribobio, China). The samples were collected, and the plates were stained using a 5-ethynyl-2′-deoxyuridine (EdU) Apollo kit (Ribobio, China) according to the manufacturer's instructions, while the cell nuclei were counterstained with Hoechst 33342. The samples were observed using a fluorescence microscope (Olympus, Japan), where statistical analysis of the images was used to calculate the percentage of EdU positive cells.

Using the In Situ Cell Death Detection Kit (Roche, Mannheim, Germany), differentially treated PC12 cells were stained for nnheim, Germany), differentially treated PC12 cells were stained for assay. To visualize cell nuclei, cells after staining were counterstained with 4′,6-diamidino-2-phenylindol (DAPI; Thermo Fisher Scientific) solution under dark at room temperature for 5 min. A fluorescence microscope (Olympus, Tokyo, Japan) was used to capture the image of fluorescence.