Coomassie brilliant blue

Chromatographic grade acetonitrile was purchased from Avantor Performance Materials, Inc. (Center Valley, PA, USA). Chromatographic grade methanol was purchased from Tianjin Shield Fine Chemicals Co., Ltd. (Tianjin, China) Analytical grade glacial acetic acid was purchased from Tianjin Fuyu Fine Chemical Co., Ltd. (Tianjin, China) The water was Wahaha pure water. Coomassie brilliant blue was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The isoquercitrin, astragalin, quercetin and kaempferol with purity greater than 98% were purchased from Chengdu Pufei De Biotech Co., Ltd. (Sichuan, China). The 4-nitrocatechol and 4-nitrophenol were purchased from A Johnson Matthey Company (Royston, UK). Phenacetin and 6β-Hydroxytestosterone and clomethiazolewere purchased from Sigma (St. Louis, MO, USA). Testosterone was purchased from Beijing J & K Technology Co., Ltd. (Beijing, China). NADPH was purchased from Blue Chemical Technology Co., Ltd. (Shanghai, China). Ketoconazole was purchased from Tokyo Chemical Industry (Tokyo, Japan).

CD45.2⁺C57BL/6 (B6) and lupus-prone MRL/lpr mice were purchased from the Shanghai Laboratory Animal Center (Chinese Academy of Sciences). CD45.1⁺ mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Animal studies were approved by the Institutional Animal Care and Use Committee of Zhongshan Hospital, Fudan University. Mice were maintained under pathogen-free conditions. Twelve-week-old MRL/lpr mice were randomized into four groups, and the mice were injected intravenously with 10 × 10⁶ ex vivo-expanded B cells or phosphate-buffered saline (PBS) control with or without 2.5 µg/g anti-IL-22 antibody (Thermo Fisher Scientific, Waltham, MA, USA) weekly for 4 weeks. The animal study is not blinding. Urine was collected for the first 24 h and assayed to detect protein by Coomassie brilliant blue according to the manufacturer’s instructions (Nanjing Jiancheng, China). Four weeks after treatment, MRL/lpr mice were sacrificed and the spleens and inguinal lymph nodes were collected and weighed. The percentages of CD4⁺IL-17⁺ Th17 cells and CD4⁺IL-22⁺ Th22 cells in the spleens were analyzed by flow cytometry, including retinoic acid–related orphan receptor γt (RORγt) and c-Maf intracellular expression. Kidney tissues were fixed for assessment.

The Ca²⁺ concentration was determined in heart tissue. Myocardial tissues were placed in a buffered solution (m/V = 1 : 9). The homogenate was centrifuged at 1000 rpm at 4°C for 10 min to remove cell debris, and the supernatant was saved for all downstream biochemical analyses. Ca²⁺ concentrations were evaluated by Coomassie Brilliant Blue staining with a commercial kit (Jiancheng, Nanjing, China).