Il 10 elisa kit

Fresh peripheral blood of humans and rats were centrifuged, the plasma was taken, and the plasma samples were stored at −20°C or −80°C for later experiments. The thawed samples were centrifuged again and then tested. According to the manufacturer's instructions, we used Neuropeptide Y (NPY) ELISA Kit (Cusabio, CSB-E08168h/CSB-E08170m), Serum T ELISA Kit (Cusabio, CSB-E05099h/CSB-E05101m), IL-1β ELISA Kit (Cusabio, CSB-E08053h/CSB-E08054m), IL-6 ELISA Kit (Cusabio, CSB-E04638h/CSB-E04639m), TNF-α ELISA Kit (Cusabio, CSB-E04740h/CSB-E04639m), IL-10 ELISA Kit (Cusabio, CSB-E04593h/CSB-E04594m), and IL-4 ELISA Kit (Cusabio, CSB-E04633h/CSB-E04634m) to detect the levels of NPY, T, IL-1β, IL-6, TNF-α, IL-10, and IL-4 in human and rat peripheral blood plasma.

After anaesthesia with sodium pentobarbital (40 mg/kg, i.p.), blood was extracted from the heart and placed in tubes containing the anticoagulant ethylenediaminetetraacetic acid (EDTA), and plasma was separated via centrifugation.
The levels of interleukin-6 (IL-6 ELISA Kit, Cusabio Biotech, CSB-E04640r, Wuhan, China), interleukin-10 (IL-10 ELISA Kit, Cusabio Biotech, CSB-E04595r, Wuhan, China), and interleukin-17A (IL-17A ELISA Kit, Cusabio Biotech, CSB-E07451r, Wuhan, China) were measured using ELISAs.
Th17 cells were labeled with CD4-FITC and IL-17A-PE; Tregs were labeled with CD4-FITC, CD25-PerCP, and FoxP3-PE. A rat peripheral blood lymphocyte isolation kit (Solarbio, P8630, Beijing, China) was used to separate lymphocytes according to the manufacturer's protocol. Then, 6 μL cocktail stimulation solution and 4 μL GolgiStop protein inhibitor were added to the lymphocytes, and the mixture was gently mixed and incubated in the dark at 37°C for 5 h. The cell surface was stained with IL-17A monoclonal antibody (Thermo Fisher Scientific, 45-7177-82, Waltham, MA, USA) or Foxp3 monoclonal antibody (Thermo Fisher Scientific, 56-5773-82, Waltham, MA, USA), and the cells were fixed and permeabilized. Intracellular staining was performed. Th17 cells and Tregs were detected via flow cytometry.

Bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF), interleukin 6 (IL-6), diethanolamine enzyme activity unit of alkaline phosphatase (ALP), transforming growth factor-β1 (TGF-β1), and interleukin 10 (IL-10) in serum were checked at 1, 2, and 4 weeks. Rat BMP-2 ELISA kit, VEGF ELISA kit, IL-6 ELISA kit, TGF-β1 ELISA, IL-10 ELISA kit (Cusabio, China), and ALP test kit (Biyuntian, China) were used. Cytokines concentration and diethanolamine enzyme activity unit were obtained according to the user's instructions.