Rk00006

For in vitro cultured Gli1⁺ cells, 1 × 10⁵ cells were cultured for 24 hours. After the medium was discarded, the cells were washed twice with preheated PBS, and MEM-α medium without serum or phenol red (Gibco) was used for further culture. One day later, the culture medium was collected for IL-1β enzyme-linked immunosorbent assay (ELISA) detection according to the manufacturer’s instructions (RK00006, ABclonal).

Plasma levels of total cholesterol (TCHO, A111-1-1), triglyceride (TG, A110-1-1), low-density lipoprotein (LDL-c, A113-1-1) and high-density lipoprotein cholesterol (HDL-c, A112-1-1), aspartate aminotransferase (AST, C010-2-1), alanine aminotransferase (ALT, C009-2-1) and creatinine (Cr, C011-2-1) were measured using commercial colorimetric enzymatic assay kit from Nanjing Jiancheng Bioengineering according to the manufacturer's instruction. Plasma TNF-α (RK00027, ABclonal, Woburn, MA), IL-1β (RK00006, ABclonal), IL-6 (Rk00008, ABclonal) and NETs (JL47089, Jonlon Biotech, Wuhan, China) levels were quantitated using relevant ELISA Kits according to the manufacturer's instructions. Human (E-EL-H1544c) and mouse (E-EL-M2435c) plasma SOST levels, as well as human (E-EL-H0080c) and mouse (E-EL-M0604c) plasma GDF 15 levels, were measured using ELISA Kit from Elabscience (Houston, TX) following the manufacturer's instructions.

The mice were sacrificed before and after SCI and spinal cord samples were collected from the injury site. Cell culture supernatants containing secreted components were collected by transferring the medium to sterile tubes, followed by centrifugation at 2000–3000 rpm for 20 min. After centrifugation, the supernatant was collected. The collected supernatant samples were then analyzed using the respective ELISA kits (RK00420, RK00027, and RK00006, ABclonal) to measure IL‐1β, IFN‐β, and TNF‐α levels. All the ELISA tests were performed in accordance with the manufacturer's instructions (ABclonal, Wuhan, China).

Proteins were extracted from mouse striatum, and the concentrations were determined for ELISA analysis. The levels of IL-1β and IL-4 proteins were measured according to the manufacturer’s instructions (RK00006, RK00036, ABclonal, China).

The mouse serum samples were diluted at 1:5 with PBS. The serum levels of IL-1β were used with colorimetric kit (RK00006, ABclonal, Woburn, MA, USA) and measured according to the manufacturer’s protocol.