Elisa kits for il10

For flow cytometry, cells were washed in PBS supplemented with 1% FCS, 1% Sodium Azide and 3 mM EDTA (FACS buffer) and stained with a cocktail of anti-CD3 PE, anti-CD4 APC-Cy7 and anti-CD25 FITC (eBioscience). Cells were then fixed and permeabilized using eBioscience FoxP3 staining kit (77-5775-40) and stained with anti-Foxp3 APC. Samples were examined using a BD LSRFortessa flow cytometer and analyzed using FlowJo software (Treestar). For ELISA, supernatants from cell culture were removed and examined for their cytokine content using ELISA kits for IL10 (DY417) and IFNγ (DY485) according to the manufacturer's instructions (R&D systems). For Western blotting, cerebellar slices were scraped from the culture membrane and suspended in PTxE buffer (PBS, 1% Triton-x, 1 mM EDTA) using mechanical homogenisation and sonication. Western blotting was performed on tissue suspensions as we have described previously [30] (link). Primary antibodies used were: anti-MOG (Millipore, MAB5680), anti-myelin basic protein (MBP) (Abcam ab40390), anti-actin (ECM Biosciences, AM2021), anti-neurofilament H (NFH) (1/1000 dilution: Millipore, MAB5539). Secondary antibodies used were: HRP conjugated mouse (Sigma, A8924), and HRP conjugated rabbit (GE Healthcare, NA934).

Cytokine secretion was evaluated to confirm the characteristics of DCs and immune status after DC treatments using enzyme-linked immunosorbent assays (ELISAs). After the differentiation of DCs, mouse blood samples were obtained and stored at − 70 °C until ELISA was performed using ELISA kits for IL-10, IL-12, and interferon (IFN)-γ (R&D Systems, Minneapolis, MN).