Mouse il 17a elisa kit

Serum was collected by terminal exsanguination of anesthetized mice and stored at −20°C. ELISA of anti-parietal (H⁺/K⁺ ATPase) autoantibody was performed from 1:10 diluted serum using the Parietal Cell ELISA kit (Fitzgerald) per manufacturer’s specifications, modified to use HRP-labeled donkey anti-mouse IgG for conjugation (Jackson ImmunoResearch Labs) diluted 1:1,000 in PBS with 0.1% BSA. ELISA of 1:2 diluted serum IL-17A concentrations was performed using the Mouse IL-17A ELISA Kit (Invitrogen) per manufacturer protocol. ELISA plates were read on a BioTek μQuant MQZ200 (BioTek) microplate reader at 450 nm. Autoantigen arrays for IgM and IgG were performed from serum samples by the University of Texas Southwestern Medical Center Microarray Core Facility using the autoantigen microarray super panel. Heatmaps were created using Morpheus open access software (Broad Institute, https://software.broadinstitute.org/morpheus). The heatmap represents relative IgG or IgM autoAb cluster (based on Ab-Score, Abs = log₂[net fluorescence intensity × signal-to-noise ratio + 1]) as provided by the University of Texas Southwestern Medical Center Microarray Core Facility. The scale of the heatmap represents the Ab-Score relative to row minimum and maximum.

2×10⁵ hUCMSCs or 1.5×10⁵ De-hUCMSCs were seeded in one well of the six-well plates. After 24 hours, cells were rinsed with PBS and 1 ml serum-free α-MEM medium (Thermofisher, Waltham, MA, USA) was added. Medium was collected 48 h later and used immediately or stored at −80°C. Colons were homogenized in PBS with 0.5% 100x Triton (Sigma, St. Louis, MO, USA) and protease inhibitor cocktail. Lysates were incubated at 4°C for 30 mins, followed by 14,000 rpm centrifuge at 4°C. Supernatant was collected and protein concentration was measured by the Bradford protein assay system (Bio-Rad). The enzyme-linked immunosorbent assay (ELISA) kits used were Mouse IL-6, IL-10 ELISA Kits (ThermoFisher Scientific, Waltham, MA, USA; EM2IL6, EM2IL10, EMTNFA), Mouse IL-17a ELISA kit (Invitrogen, Carlsbad, CA, USA; KMC3021), Prostaglandin E₂ EIA Kit-Monoclonal (Cayman, Ann Arbor, MI, USA; 514010) and Human IL-2, IL-10 (ThermoFisher Scientific; EH2IL2, EHIL10).