Thrombin

The levels of thrombin (Abcam), thrombin‐antithrombin complexes (TAT), interleukin‐4 (IL‐4), IL‐5, IL‐13 in BALF and total serum IgE (Elabscience) were measured by Enzyme‐linked immunosorbent assay (ELISA) kits, respectively. The protocols were followed according to the manufacturer's instructions. Briefly, the standard working solution and the sample to be tested were separately added to a 96‐well plate at 100 μL per well. One hundred microliter of biotin‐conjugated antibody was added to each well and incubated at 37°C for 1 hour. Next, 100 μL of the enzyme conjugate working solution was added per well and incubated at 37°C for 30 minutes. Subsequently, a substrate solution (TMB) was added to each well at 90 μL per well and incubated at 37°C for 15 minutes in the dark. Finally, 50 μL of the stop solution was added to each well to terminate the reaction. The optical density (OD) of each well was measured using a microplate reader set to 450 nm. A standard curve of the average of the OD repeat readings is created to calculate the concentration to be tested.

The primary cultured astrocytes were harvested after treatment with various drugs for different time. As for the measurement of MIF in the cord tissue samples, the contusion model was established as mentioned above, and the PAR1 inhibitor SCH79797 or vehicle was injected intrathecally before the incision was sutured. Spinal cord samples (n = 6 in each time point) at 1 cm length around the lesion site were harvested on 0 day, 1 day, 4 days, and 1 week after the rats were anesthetized with 10% chloral hydrate (300 mg/kg) and transcardially perfused with saline. Each sample weighed about 0.03–0.04 g. The cells or tissue samples were sonicated using the lysis buffer supplemented with a protease inhibitor PMSF. Homogenate was centrifuged at 12,000 rpm for 15 min at 4 ℃, and the supernatant was collected for MIF (Elabscience) and thrombin (Elabscience) ELISA assay. The concentrations of MIF and thrombin are expressed as ng/ml for the supernatant, while ng/mg for the cord tissues. Plates were read with a multifunctional enzyme marker (Biotek Synergy2) at a 450 nm wavelength.

Protein samples were prepared using the extraction buffer supplemented with a protease inhibitor PMSF as mentioned above. Homogenate was centrifuged at 12,000 rpm for 15 min at 4 °C, and the supernatant was collected for TNF-α, IL-1β, IL-6, IL-4, IL-10 (MULTI SCIENCES), and thrombin (Elabscience) ELISA assay. The contents of the cytokines were expressed as pg/ml/10⁵ cells for the supernatant, and pg/mg for the lysate of the cells and the cord tissues. Plates were read with a multifunctional enzyme marker (Biotek Synergy2) at a 450-nm wavelength.