Ab208979

Serum sampling was performed on mice after they received an intraperitoneal injection of 150 mg/kg pentobarbital sodium. Serum samples were intraperitoneally collected from mice after 12-h fasting and analyzed using a microplate reader (Thermo Fisher, Waltham, MA, USA) at 450 nm based on enzyme-linked immunosorbent assay (ELISA) to obtain the concentration of cytokines, including MCP-1 (ab208979; Abcam, Waltham, MA, USA), IL-1β (ab197742; Abcam, Waltham, MA, USA), IL-6 (ab222503; Abcam, Waltham, MA, USA), and IL-17 (ab199081; Abcam, Waltham, MA, USA).

The serum of humans and mice and the supernatant of HK-2 cells were collected. ELISA kits were used to detect the levels of blood urea nitrogen (BUN) (YS02947B, Jiya biological and technology co., Ltd, Shanghai, China), SCr (FT-P9S3176X, Fantai biological and technology co., Ltd, Shanghai, China), tumor necrosis factor (TNF)-α (ab285312, ab285312, Abcam), interleukin (IL)-1β (ab214025, ab255730, Abcam), IL-6 (ab178013, ab222503, Abcam), and monocyte chemoattractant protein (MCP)-1 (ab179886, ab208979, Abcam) in the serum and cell supernatant.

Colonic tissue homogenate was prepared using a radio-immunoprecipitation assay (RIPA) buffer and centrifuged at 12,000 g and 4°C for 10 min to obtain the supernatant. Next, the cells were centrifuged at 1,000 g for 10 min to collect the supernatant. The concentration of tumor necrosis factor-α (TNF-α) (ab208348, Abcam), IL-1β (ab197742, Abcam), monocyte chemoattractant protein-1 (MCP-1) (ab208979, Abcam), and IL-10 (ab255729, Abcam) were subsequently determined using ELISA kits. Furthermore, the absorbance at a wavelength of 450 nm was determined using a microplate reader (Bio-Rad) [25 (link)].

In cultured cells, GFP was detected by direct fluorescence. The mouse spine tissue was fixed with formalin and embedded in paraffin. Thin sections of the tissue were then cut using a microtome and placed onto glass slides. The sections are then deparaffinized and rehydrated using a series of alcohol solutions. The sections are then stained with hematoxylin, which stains the nuclei of the cells blue, and eosin, which stains the cytoplasm and extracellular matrix pink. The slides are then dehydrated again and coverslipped. Enzyme-linked immunosorbent assay (ELISA) was performed to measure the levels of mouse Ccl2 (ab208979; Abcam, Hangzhou, China), mouse Il1β (ab197742; Abcam), mouse tumor necrosis factor alpha (TNFα, ab208348; Abcam), mouse interferon gamma (IFNɣ, ab282874; Abcam), mouse Il6 (ab100712, Abcam), mouse Il17 (ab100702; Abcam), mouse arginase 1 (ARG1, ab269541; Abcam), mouse CD163 (ab272204, Abcam), mouse transforming growth factor β1 (TGFβ1, ab119557, Abcam) and mouse Il10 (ab108870, Abcam) in cell lysis. Specific kits were utilized as per the manufacturer’s instructions.