Mouse cirp elisa kit

RAW 264.7 cells were cultured in Opti-MEM (Thermo Fisher Scientific) and pre-treated with GW4869 (MilliporeSigma) at a dose of 10 μg/ml for 2 h followed by 20 h stimulation with 1 μg/ml LPS. An equal volume of the supernatants was supplemented with Lane Marker Non-Reducing Sample Buffer (Thermo Fisher Scientific) to perform Western blotting for CIRP. LPS at a dose of 5 mg/kg was intraperitoneally injected to WT mice with or without 1 mg/kg GW4869 injection simultaneously. Serum was harvested 4 h after the injection and eCIRP levels of the serum were assessed by mouse CIRP ELISA Kit (LSBio). CLP was performed in WT mice with or without intraperitoneal instillation of GW4869 before closing the abdomen. 20 h after CLP, serum was harvested to assess eCIRP levels by mouse CIRP ELISA Kit (LSBio).

Exosomes isolated from the serum of LPS-injected or CLP mice were lysed using extraction buffer containing 25 mM Tris, 0.15 M NaCl, 1 mM EDTA, 1% NP-40, 5% glycerol, 2 mM Na₃VO₄, and protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland), at pH 7.4. Protein concentration was determined by the Bio-Rad protein assay reagent. CIRP levels of exosomal lysates were assessed using mouse CIRP ELISA Kit (LSBio, Seattle, WA) and normalized by protein concentrations.