Splenic concentrations of TNF-α, IL-1β and IL-10 were measured by ELISA using commercially available kits, including a mouse TNF-α ELISA kit (cat. no. 558534, BD Biosciences), a mouse IL-1β ELISA kit (cat. no. MBS824757) and a mouse IL-10 ELISA kit (cat. no. MBS8244590; both MyBioSource, Inc., San Diego, CA, USA), respectively, as previously described (18 (link)). Tissue samples (10–15 mg) were homogenized in a tissue grinder containing 1 ml lysis buffer (PBS containing 2 mM phenylmethylsulfonyl fluoride and 1 mg/ml aprotinin, leupeptin, and pepstatin A, all obtained from Sigma-Aldrich; Merck KGaA), as described by Clark et al (21 (link)). Analysis was performed with 100 ml standard (diluted in lysis buffer) or 10, 50 or 100 ml tissue homogenate. Each sample was run in duplicate and a portion of the sample was analyzed for protein. Data were expressed as pg/mg of protein. For each assay, a standard curve was generated and based on replicates of the measured absorbance, demonstrated an average coefficient of variance of <10%.