Legend max mouse tnf α elisa kit

Lungs were isolated from neonatal mice following retrograde vascular perfusion. Isolated lungs were flash frozen in liquid nitrogen and stored at -80 ^oC until processed further for cytokine analysis. Briefly, lungs were homogenized in 1 ml of Tissue Protein Extract Reagent (T-PER) (Life Technologies, Grand Island, NY) containing protease inhibitor cocktail (Thermo Scientific, Waltham, MA). Homogenates were centrifuged at 10,000xg for 5 min and supernatants were collected for analysis. IL10 levels were determined by mouse IL10 quantikine ELISA kit following manufacturer’s (R&D systems, Minneapolis, MN) instructions. TNFα levels were measured using Legend Max mouse TNFα ELISA kit (BioLegend, San Diego, CA) following manufacturer’s instructions. Data was normalized to the total protein content in the supernatants as determined by BCA protein assay (Life Technologies, Grand Island, NY).

Blood was collected from the orbital vein to non-heparinized tubes and let to clot and spun at high speed to collect serum. Serum levels of SAA3, TNFα and CTX-I were measured using the mouse SAA-3 ELISA kit (Sigma-Aldrich), LEGEND MAX™ Mouse TNF-α ELISA Kit (BioLegend) and the RatLapsTM EIA CTX-I (Immunodiagnostic Systems, Denmark), following the manufacturer’s instruction.
Human peripheral blood was collected by venous puncture and plasma was recovered. SAA and CTX-I were measured using the human SAA 1 and 2 ELISA (Sigma-Aldrich) and Serum Crosslaps (CTX-I) ELISA (ImmunoDiagnosticSystems, Denmark), following the manufacturer’s instruction. Circulating levels of cytokines/chemokines and bone biomarkers were determined using the Human Cytokine/Chemokine Panel A 48-Plex Discovery Assay^® Array (HD48A) and Human Bone 13-Plex Discovery Assay^® Array (HDBN13), respectively (Eve Technologies, Calgary, Canada).

Expression of TNF-α and IFN-γ cytokines was measured on CD4⁺ T cell supernatants using enzyme-linked immunosorbent assay (ELISA), LEGEND MAX™ Mouse TNF-α ELISA Kit and LEGEND MAX™ Mouse IFN-γ ELISA Kits (BioLegend), and following the manufacturer’s instructions. IL-6 expression was measured from PyMt culture media after treatment using the LEGEND MAX™ Mouse IL-6 ELISA Kit (BioLegend). The same amount of total protein, quantified using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific), was used for the assays. Optical densities were measured on a Synergy Neo2 (Biotek, Winooski, VT, United States) at 450 nm and cytokine concentrations were calculated with a five-parameter logistic curve using Gen5 Microplate Reader and Imager Software (Biotek).