Mouse il 10 duoset elisa kit

Mice MSCs were isolated from the femurs and tibias by flushing the bone marrow cells. 18 The connective tissue around the bones was removed, then the bone marrow plugs were flushed using Dulbecco's modified Eagle's medium supplemented with 15% fetal bovine serum (FBS), 100 units/ml penicillin G, and 0.1 mg/ml streptomycin. The bone cavities were repeatedly flushed to obtain enough marrow cells. The cells were then plated and cultured in the same media at 37°C with 5% CO 2 . The medium was changed the next day and the non-adherent cells were removed. Medium was replaced every 3 days and the cells were cultured until confluency exceeded 90%. Morphology of MSCS was examined by phase contrast microscopy using a Cytation 5 imaging reader (BioTek, USA). In some experiments, MSCs were incubated with 1 mM 2-deoxyglucose (2-DG) for 24 h to block glycolysis and the levels of IL-10 and CTLA-4 determined. Glycolysis Assay kit (Extracellular acidification, Catalog no. ab197244) was obtained from abcam (Toronto, ON). Mouse IL-10 DuoSet ELISA kit (Catalog no. DY417-05) was obtained from R&D Systems (Minneapolis, MN). CTLA-4 Mouse ELISA kit (Catalog no. EMCTLA4) was obtained from Thermo Fisher (Winnipeg, MB). Unless otherwise indicated, all other reagents used were of analytical grade and were obtained from either Thermo Fisher Scientific (Winnipeg, MB) or Sigma-Aldrich (Oakville, ON).

For the detection of cytokines in the cell culture supernatants or sera, ELISAs were performed as described previously (17) , using the primary capture mAbs anti-TNFα, anti-IL-6, and anti-IL-1β and their corresponding biotinylated detection mAbs (Biolegend, San Diego, CA). Other ELISA reagents included: recombinant mouse TNFα, IL-6, and IL-1β as standards (Biolegend, San Diego, CA), HRP-conjugated avidin D (Vector Laboratories, Burlingame, CA), and TMB microwell peroxidase substrate and stop solution (Kirkegaard and Perry Laboratories, Gaithersburg, MD). IL-10 cytokine was measured using Mouse IL-10 DuoSet ELISA kit (R&D Systems, Minneapolis, MN) according to manufacturer's instructions. Biological replicates were used, representing independent BMDM cultures from individual mice.