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Our animal model demonstrates the crucial role of CXCR2 ligands/CXCR2 in acute lung inflammation and injury as a result of intratracheal dsRNA.MethodsReagents RNA instillation: Double-stranded RNA (dsRNA, Poly IC) and single-stranded RNA (ssRNA, Poly C) have been purchased from Sigma-Aldrich Corp. (St. Louis, Mo.) and reconstituted in sterile normal saline (20 / ) and stored at four before use. Enzyme-linked immunoadsorption assay (ELISA) experiments Capture and Detection antibodies to murine KC/CXCL1 and murine MIP-2/CXCL2/3 had been purchased as DuoSetfrom R D Systems (Minneapolis, MN).Neutralization research: Purified rat anti-mouse Ly-6G (Gr1) mAb (clone RB6-8C5) was bought from BD Pharmingen (San Diego, CA) and was used for neutrophil depletion research as previously described [24]. Polyclonal goat anti-murine CXCR2 was made by the immunization of a goat having a peptide containing the ligand-binding sequence Met-Gly-Glu-Phe-Lys-Val-Asp-Lys-Phe-AsnIle-Glu-Asp-Phe-Phe-Ser-Gly of CXCR2 [24-31]. The goat was immunized with CXCR2 in various intradermal web-sites with full Freund's adjuvant (CFA) followed by no less than 3 boosts of CXCR2 in incomplete Freund's adjuvant (IFA) as previously described. [24-31]. Direct ELISA was applied to evaluate antisera titers, and sera was utilised for Western blot, ELISA and neutralization assays when titers had reached greater than 1/1,000,000. The CXCR2 protein sequence has been shown to contain the ligand-binding , S182, 1660 South Columbian Way, Seattle, WA 98108, USA Full list of author portion with the CXCR2 receptor [24-26,32]. The antiCXCR2 antibodies have been employed previously to block mouse CXCR2 in vivo, and has been shown to detect CXCR2 by Western blot and fluorescence-activated cell sorting evaluation of neutrophils in vivo [24-26,32]. The anti-CXCR2 antibody has been shown to be neutralizing employing both in vitro neutrophil chemotaxis assay and in vivo by abrogating the influx of neutrophils into the peritoneum of standard mice in response to exogenous ELR-positive murine CXC chemokines [24-26,32]. In vivoPage two of(web page quantity not for citation purposes)Journal of Inflammation 2005, two:http://www.journal-inflammation.com/content/2/1/administration of anti-CXCR2 antibodies inhibited pulmonary neutrophil sequestration in murine models of Aspergillosis, Nocardia, and Pseudomonas pneumonia and prevented the influx of neutrophils in urine plus the kidney in a murine model of Escherichia coli urinary tract infection [24-26,32]. In addition, intraperitoneal administration of this antibody did not alter peripheral blood neutrophil counts [24-26,32]. 1 ml of antiserum against mCXCR2 and control antibody is approximately 10 mg of IgG.Murine model of dsRNA-induced lung injury We employed six week old female BALB/c mice to intratracheally inject either single-stranded (ssRNA) or doublestranded RNA (dsRNA) using a modification of a previously described technique [30,33-36]. Mice had been anesthetized with ketamine (60 mg/kg) intraperitoneally; then, below sterile circumstances, the anterior neck soft tissue was dissected to expose the trachea and 50 RNA (20 / ; 40 /g mouse wt) was injected via 26 gauge tuberculin needle and syringe attached to a Steppermicroinjector (Indicon, Inc., Brookfield, CT) into the trachea beneath direct visualization. Immediately following the instillation, the skin was apposed and closed making use of tissue Verse species ranging from yeast to humans. The mammalian SUT-2 protein adhesive and also the mice were permitted to recover from anesthesia before replacement into their cages.Immunolocalization of TLR3.