Krebs ringer buffer

To induce hyperuricemia, oteracil potassium (OP) (Meilunbio, Dalian, China), yeast extract (Oxoid, Hampshire, UK), and uric acid (Shanghai Yuanye Biotechnology, Shanghai, China) were purchased. Benzbromarone was from Excella GmbH (Feucht, Germany). Febuxostat was from TCI (Tokyo, Japan). SHR4640 and compound CC18002 were synthesized by Professor Zhiyan Xiao’s lab at the Institute of Materia Medica, CAMS & PUMC. For the measurement of in vivo experimental parameters, the kit for detecting blood uric acid was purchased from Biosino (Beijing, China), and the kit to determine plasma XOR activity was from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). For the in vitro experiment, Krebs-Ringer buffer (Solarbio, Beijing, China) and uric acid kits (abcam, Cambridge, UK) were purchased. Milk-derived XOR, xanthine, and DMSO were purchased from Sigma-Aldrich (Darmstadt, Germany).

Intracellular Ca²⁺ concentration was examined with Fluo-3 AM (Abcam, USA). Cells were seeded in 12-well plates and incubated with 2 μM Fluo-3 AM diluted in Krebs–Ringer buffer (Solarbio, Beijing, China) for 30 min. After removing the culture and washing with Krebs–Ringer buffer, the cells were collected and rinsed by PBS without Ca²⁺. Finally, the cells were measured by flow cytometry (Beckman Coulter, USA).