Elisa kits for tumor necrosis factor α tnf α

Enzyme-linked immunosorbant assays (ELISAs) were performed to quantify cytokines in culture supernatants (media) from hBMSCs exposed to BCM for 1 and 3 days. ELISA kits for Tumor necrosis factor α (TNF-α) (R&D Systems, Minneapolis, MN), Interleukin-6 (IL-6) (R&D Systems, Minneapolis, MN), stromal cell derived factor 1a (SDF-1) (TSZ ELISA^TM, Framingham, MA), vascular endothelial growth factor (VEGF) (Quantikine®, Minneapolis, MN), and human cathelicidin LL-37 (Hycult® Biotech, Netherlands) were performed according to the manufacturer’s instructions. Briefly, supernatants (media) were collected from individual wells containing hBMSCs exposed to BCM at days 1 and 3 (0.5 mL volumes), treated with protease inhibitor cocktail (Sigma-Aldrich, St. Louis MO), and stored at −80°C until analysis. Total protein in samples was quantified using a BCA assay (Pierce). Cytokine levels in supernatants were quantified by extrapolation from standard curves generated using recombinant protein for each respective cytokine, and determined concentrations were normalized to total protein content in each supernatant sample as determined by the BCA assay.

The ISO was procured from Sigma (St. Louis, MO, USA). The TAX was purchased from Biosynth Carbosynth (Berkshire, UK). A Cardiac troponin I (cTnI) ELISA kit was obtained from Kamiya (Tukwila, WA, USA). Kits for creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) activities were procured from Spinreact (Girona, Spain). A HO-1 ELISA kit was obtained from MyBioSource (San Diego, CA, USA). The ELISA kits for tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were supplied by R&D Systems (Minneapolis, MN, USA). Anti-NF-κB p65, anti-caspase-3, and anti-Nrf2 antibodies were obtained from ThermoFisher (Waltham, MA, USA; 1:100 dilution), while anti-Bax and anti-Bcl-2 antibodies were supplied by Abcam (Cambridge, MA, USA; 1:100 dilution).