Il 4 elisa kit

TNFα and IL-6 cytokines in the supernatant of microglia culture were measured using ELISA kits purchased from BioLegend (San Diego, CA). Cerebellum and lymph node samples were homogenized in 0.5 mL of ice-cold lysis buffer (Cell Signaling Technology, Beverly, MA) supplemented with protease inhibitors (Roche Diagnosis, Mannheim, Germany) by rapid agitation for 2 min in the presence of 3-mm stainless beads. The tissue lysate was centrifuged for 10 min at 13,000×g in a cold microfuge, and the supernatant was transferred to a new tube. The concentration of proteins in the lysate was determined by Bradford protein assay. The tissue levels of IL-4 were determined using a high sensitivity IL-4 ELISA Kit (eBioscience, San Diego, CA), and the concentration of IL-4 in serum samples was quantified using Mouse IL-4 DuoSet (R&D Systems), according to the manufacturer’s instruction.

IFNγ and IL-4 cytokines in the supernatant of activated CD4⁺ T cell culture and tissue lysates were measured using ELISA kits as previously described [19 (link)]. Briefly, lymph node, spinal cord and cerebellum tissues were homogenized in lysis buffer supplemented with protease inhibitors (Cell Signaling Technology, Danvers, MA, USA) by rapid agitation using 3-mm stainless beads and a TissueLyser II (Qiagen, Hilden, Germany) homogenizer for 2 min. The levels of IL-4 in tissue lysates were determined using a high sensitivity IL-4 ELISA Kit (eBioscience, San Diego, CA, USA) according to the manufacturer’s instruction. The amount of IFNγ was determined using IFNγ kit purchased from PeproTech (Rocky Hill, NJ, USA).