E el r0016c

On the 3rd day, after HRV monitoring, 6 rats from each group were anesthetized with isoflurane inhalation [17 (link), 18 (link)]; then, the chest cavity was dissected, and 2 mL of blood was taken from the right atrium for ELISA and 2 mL for flow cytometry (FCM). For ELISA, blood was preserved at room temperature for 2 h prior to centrifugation at 3000 r/min at 4°C for 10 min; then, the upper layer of the serum was separated and stored in the refrigerator at -80°C for detection. For FCM, fresh blood was taken for testing. According to the manufacturer's instructions [19 (link)], the serum levels of TNF-α, IL-1α, IL-10, IL-6, HMGB1, and sCD14 were determined with ELISA.
The main related antibodies include: rat anti-TNF-α (lot number: E-EL-R0019c, manufacturer: Elabscience), rat anti-IL-1α (lot number: E-EL-R0011c, manufacturer: Elabscience), rat anti-IL-6 (lot number: E-EL-R0015c, manufacturer: Elabscience), rat anti-IL-10 (lot number: E-EL-R0016c, manufacturer: Elabscience), rat anti-HMGB-1 (lot number: E-EL-R0505c, manufacturer: Elabscience), and rat anti-sCD14 (lot number: CSB-E11178r, manufacturer: Cusabio).

After modeling (9 days after surgery), the whole blood and hippocampus tissues were harvested. After 2 h of standing, the whole blood was centrifuged for 3 min at 9,000 rpm. The serum was then collected to analyze the expression of cytokines. The hippocampus tissues were homogenized, and the levels of IL-1β (Elabscience, E-EL-R0012c), IL-6 (Elabscience, E-EL-R0015c), IL-12 (Elabscience, E-EL-R0064c), tumor necrosis factor (TNF)-α (Elabscience, E-EL-R2856c), IL-18 (Elabscience, E-EL-R0567c), colony-stimulating factor (CSF)-1 (Elabscience, E-EL-R0601c), IL-4 (Elabscience, E-EL-R0014c), IL-10 (Elabscience, E-EL-R0016c), transforming growth factor (TGF)-β (Elabscience, E-EL-0162c), and IL-13 (Elabscience, E-EL-R0563c) were determined according to the manufacturers’ instructions.

The ELISA kits were used to evaluate the expression levels of anti‐inflammatory cytokines including TGF‐β (E‐EL‐R1015c; Elabscience) and IL‐10 (E‐EL‐R0016c; Elabscience) and pro‐inflammatory cytokines including TNF‐α (E‐EL‐R2856c; Elabscience) and IL‐1β (E‐EL‐R0012c; Elabscience) in the injured spinal cord tissue. On the 7th day after SCI, the hearts of rats were perfused with 0.9% normal saline, and then the spinal segments around the lesion center were removed. The spinal cord tissue was cut into 1 mm³ piece, and pre‐chilled RIPA protein lysate containing PMSF was added to grind into homogenates in liquid nitrogen. The nucleic acid was sonicated and lysed at 4°C for 20 min. The total protein was extracted by centrifugation at 12,000×g for 5 min. The supernatant was taken, and protein quantification was performed by BCA. The ELISA kits were used to detect the expression levels of TGF‐β and IL‐10 (anti‐inflammatory cytokines) and TNF‐α and IL‐1β (pro‐inflammatory cytokines) according to the manufacturer's protocol. The optical density values were measured with a microplate reader (Multiskan MK3; Thermo Fisher Scientific), and the corresponding cytokine concentrations were calculated from the standard curve.