Mouse il 33 duoset elisa

IL-33 levels were measured using Mouse IL-33 Duo Set ELISA (R&D systems, #DY3626) following manufacturers protocol. For determination of IL-33 levels by VAT DCs, 2x10⁴ sorted cells from lean and obese mice were seeded in 96 wells for 24 h and media were collected.

Fecal lipocalin-2 levels in distal colon fecal contents were analyzed using Mouse Lipocalin-2 DuoSet ELISA (R&D Systems, DY1857) according to manufacturer-provided instructions. IL-13 and IL-33 levels in distal colonic epithelial scrapings or IL-33 in conditioned media from cultures of peeled distal colonic epithelium were analyzed using Mouse IL-13 DuoSet ELISA (R&D Systems, DY413) or Mouse IL-33 DuoSet ELISA (R&D Systems, DY3626) according to manufacturer instructions.

The mice were intubated and subjected to bronchoalveolar lavage (BAL) with 0.6 mL normal saline three times through a tracheal cannula. Total cell counts in the BALF were calculated using a hemocytometer. Red blood cells were removed using the Red Blood Cell Lysing Buffer Hybri-Max (Sigma-Aldrich). Cytospun BALF was stained with Diff-Quik Reagent (Sysmex, Kobe, Japan), and differential cell counts were calculated by counting > 400 cells per mouse using a microscope. After performing the BAL, the lungs were inflated with the Tissue-Tek O.C.T. Compound (Sakura Finetek Japan, Tokyo, Japan) (50% vol/vol) diluted with PBS containing 10% sucrose and stored at -80 °C. Proteins in the BALF were assessed using enzyme-linked immunosorbent assay (ELISA): interleukin (IL)-33 (Mouse IL-33 DuoSet ELISA; R&D Systems, Minneapolis, CA, USA) and MMP-12 (Mouse MMP-12 Kit Price PicoKine; BOSTER Biological Technology, Pleasanton, CA, USA).