Edu 488 cell proliferation detection kit

For MTT assay, 1 × 10³ cells were seeded into the 96-well plate. The cells were grouped in a time gradient and treated with MTT for 2 h, then the medium was removed and 200 μL DMSO was added for incubation. The 560 nm absorbency was measured by microplate reader.
For EdU assay, 2 × 10⁴ cells were seeded into the 24-well plate. We used the EdU-488 Cell Proliferation Detection Kit (Beyotime: China) for experiments.

The proliferation of HUVECs was detected by the EdU-488 Cell Proliferation Detection Kit (Beyotime, Shanghai, China). The HUVECs were incubated with the EdU solution for 2 h in an incubator with 5% CO₂ at 37 °C. The cells were fixed and stained with Azide-fluor 488. The nuclear counterstaining was carried out by Hoechst33342. Photos were taken with ImageXpress Confocal HT.ai (Molecular Devices, San Jose, CA, USA).

To examine cell proliferation, EdU (50 mg/kg body weight, Beyotime, China) was intraperitoneally administered to each animal every 2 days over the 8-day period, initiated 24 h after MCAO/r surgery. EdU staining was performed with the EdU-488 cell proliferation detection kit (Beyotime, China). Briefly, frozen slides were sequentially incubated with pre-configured click reaction reagent (click reaction buffer: CuSO4: azide 488: click additive solution = 430: 20: 1: 50). TUNEL staining was performed to measure apoptosis with the TUNEL apoptosis assay kit according to manufacturer protocol (Beyotime, China). Briefly, frozen slides were sequentially incubated with pre-configured TUNEL detection reagent (terminal deoxynucleotidyl transferase: FITC–dUTP = 1: 9). The apoptosis index is calculated by dividing the number of TUNEL-positive cells by the total number of cells per-field of view. The fluorescence signal was detected by laser confocal microscope (Olympus Optical, Ltd., Japan). For TTC staining, brain tissues were cut into six sections in the coronal plane (2 mm), and then dipped in 2% TTC (Solarbio, China) for 30 min at 37 °C. The infarcted volume was measured and presented as a percentage of the non-ischemic hemisphere to correct for edema.