Accuskan fc filter based microplate photometer

Enzyme activity experiments were performed on a sample of nine rats (4 SHAM, 5 HF‐rEF). A tissue sample (~180 mg) from the white portion of the gastrocnemius muscle was collected from the right hindlimb. Samples were rinsed with 0.1 M TRIS‐Buffer (pH 7.4), and homogenized in 1 ml of 0.1 M TRIS‐HCl, pH 7.8, containing 1 mM EDTA for 1 min at 5 m/s using 2 ml tubes containing ~0.5 g of 1.4 mm ceramic beads using Bead Mill 4 (Fisherbrand^™). Samples were then centrifuged at 10,000g for 15 min at 4℃. The supernatant was removed and analyzed for COX‐2 activity (Cayman Chemical; Item no. 760151) according to manufacturer's instructions. Absorbance was read at 590 nm using accuSkan^™ FC Filter‐Based Microplate Photometer (Fisherbrand^™).

IL-17A production by bovine PBMCs was determined using the Bovine IL-17A Do-It-Yourself ELISA kit (Kingfisher Biotech, Inc) following the manufacturer's published ELISA Technical Guide (Kingfisher Biotech, Inc). In summary, anti-bovine IL-17A capture antibody was coated overnight at 4°C in 96-well plates (Themo Scientific MaxiSorp, Immulon 4 HBX) at a concentration of 2.5 μg/ml. Plates were then blocked with a 4% bovine serum albumin in PBS solution for 1 h at room temperature. Plates were washed with a 0.05% Tween 20 in PBS buffer, and bovine IL-17A standard or stimulated PBMC cell culture supernatants (from the MTT cell viability assays) were incubated at room temperature for 2 h. The plates were washed and then incubated with biotinylated anti-bovine IL-17A detection antibody at a concentration of 0.1 μg/ml for 1 h at room temperature. The plates were washed and then incubated with 1:100 HRP-Streptavidin (Kingfisher Biotech) in assay diluent for an additional hour at room temperature. Plates were washed a final time, and then incubated with 1-Step Ultra TMB ELISA Substrate (ThermoFisher Scientific) until sufficient color change was detected in the standards. The reaction was then stopped with 2 M H₂SO₄ stop solution and the sample absorbance was measured at 450 nm using an ELISA microplate reader (Fisher Scientific accuSkan™ FC Filter-Based Microplate Photometer).