Edu imaging kits

Cell climbing was placed in the 24-well plate, and 2 × 10⁵ cells were inoculated into the 24-well plate and cultured overnight. 2 hours before harvest, we added EdU solution (10 μM/ml) and incubated for 20 minutes. In accordance with the manufacturer's directions, EdU Imaging Kits (K1075, Apexbio) was used to stain the proliferative cells.

Maresin‐1 and Maresin‐1 ELISA kit were purchased from Cayman Chemical. AngII was purchased from Enzo Life Sciences. Mouse IL‐1β ELISA kit was purchased from Neobioscience. EdU imaging kits was purchased from APExBIO. ML385 and KN‐93 were purchase from Topscience. Fluo‐4 AM was purchased from Invitrogen. Fetal bovine serum was purchased from Inner Mongolia Opcel Biotechnology Co., Ltd. Primary antibodies included: anti‐GAPDH (Abcolonal), anti‐SM22α (Servicebio), anti‐ASC (Santa Cruz Biotechnology), anti‐caspase‐1 p20 (Santa Cruz Biotechnology), anti‐IL‐1β (Santa Cruz Biotechnology), anti‐OPN (Immunoway), anti‐NLRP3 (Immunoway), anti‐IL‐18 (ImmunoWay), anti‐GSDMD‐N (Immunoway), anti‐Nrf2 (GeneTex), anti‐α‐SMA(Abcam), anti‐HO‐1(Abcam), anti‐CaMKII (Abcam), anti‐p‐CaMKII (Zenbio), anti‐PCNA (Cell Signaling Technology), anti‐rabbit IgG (H+L) (Cell Signaling Technology), anti‐mouse IgG (H+L) (Cell Signaling Technology), Cy3‐conjugated goat anti‐rabbit IgG (H+L) (Servicebio), and Alexa Fluor® 488‐conjugated goat anti‐rabbit IgG (H+L) (Servicebio).

We used EdU Imaging Kits (APExBIO, Houston, TX, USA) based on the manufacturer’s instruction to perform this assay. The cells were plated in a 20 mm glass bottom cell culture dish and exposed to 10 μmol/L EdU for 4 hours. Following this, the cells were fixed at room temperature for 15 minutes with 4% paraformaldehyde in PBS followed by 0.5% Triton-100 in PBS. The click reaction solution, as configured, was subsequently introduced and incubated for 30 minutes at room temperature in dark conditions. Following three washes with PBS (3 minutes each), the cells were then incubated with a diluted solution of Hoechst 33342 (1:2000) at room temperature in the dark for 30 minutes. The images were obtained under laser confocal microscopy (Olympus, Tokyo, Japan), and the number of positive cells (red stained) was counted using ImageJ.