2.3 mm zirconium beads

A 100 mg aliquot of each frozen dry fecal specimen was resuspended in 1 ml of extraction buffer [phosphate buffered saline (PBS, pH 7.4) containing 0.01% soybean trypsin inhibitor, 0.1% ethylenediaminetetraacetic acid, and 0.05% Tween 20, all from Sigma, St. Louis, MO] and ~1.5 g of 2.3 mm zirconium beads (Biospec, Bartlesville, OK) were incorporated. Fecal samples were subjected to 1-min beating cycle in a mini-beadbeater-8 tissue homogenizer (Biospec, Bartlesville, OK) and centrifuged at 14,000 rpm for 1h at 4°C. The supernatant was collected and stored at -80°C until use. IL-1β, IL-1α, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p40/IL-23, IL-12p70, IL-15, IL-16, IL-17, Eotaxin, Eotaxin-2, Eotaxin-3, IP-10, MCP-1, MCP-2, MCP-3, MCP-4, MDC, MIP-1α, MIP-1β, TARC, GM-CSF, TNF-β, VEGF-A, and IFN-γ were quantified in fecal supernatants using human multiplex immunoassays [Meso Scale Discovery (MSD), Rockville, MD] according to the manufacturer’s protocol. Each fecal lysate was tested in duplicate. Plates were read using the QuickPlex SQ 120 (MSD, Rockville, MD). The concentration of each analyte was determined using standard calibrators and analyzed using the Meso Scale Discovery Workbench software v15.0 (MSD, Rockville, MD).

A 300-mg aliquot of each stool sample was placed in preweighed tubes containing ~1.5 g of 2.3-mm zirconium beads (Biospec, Bartlesville, OK) and 1 mL of extraction buffer (phosphate-buffered saline [PBS; pH 7.4] containing 0.01% soybean trypsin inhibitor, 0.1% EDTA, 0.5% phenylmethanesulfonyl fluoride solution, and 0.05% Tween 20, all from Sigma, St. Louis, MO). Stool samples were subjected to three 1-min beating cycles in a mini-Beadbeater-8 tissue homogenizer (Biospec, Bartlesville, OK) with 2-min incubations on ice between cycles and centrifuged at 14,000 rpm for 30 min at 4°C. The supernatant was collected, 10 μL of 1% bovine serum albumin containing 0.1% sodium azide (vol/vol) (Sigma) was added, and the mixture was stored at −80°C until use.