Asys expert plus

Interleukin (IL)-6 and IL-8 levels in supernatant cultures were evaluated using a commercial antibody-specific ELISA kit (Peprotech, East Windsor, NJ, USA), according to the manufacturer’s instructions. For both ShCTRL and ShTWIST cells, four different conditions were analyzed: (1) nontreated cells, (2) incubation with human recombinant IL-17A (50 ng/mL, Peprotech), (3) incubation with IL-17F (50 ng/mL, Peprotech), and (4) incubation with IL-17A and IL-17F simultaneously (25 ng/mL each). The experiments were performed in triplicate and analyzed with an ELISA microplate reader at 450 nm (Asys Expert Plus, Biochrom, Cambridge, UK). The results are represented as the ratio of the experiments over the control.

Measurements of the level of serum amyloid A in blood serum and uterine flush was performed using a commercial ELISA kit (Tridelta Development Ltd., Maynooth, Kildare, Ireland). The inter- and intra-assay coefficients of variation for SAA analysis were <12.1 and <7.5 %, respectively. The determination of haptoglobin in blood serum and uterine flush was performed using a commercial colorimetric assay kit (Tridelta Development Ltd., Kildare, Ireland). The inter- and intra-assay CVs for Hp analysis were <5.7 and <6.3 %, respectively. Procedures were performed according to the manufacturers’ instructions and literature methods (Suojala et al. 2008 (link) and Tothova et al. 2012 (link)). Absorbance readings and subsequent calculations of final concentrations were performed on an automatic microplate reader (Asys Expert Plus, Biochrom Ltd., Cambridge, England) at 450 nm, 630 nm for Hp, and 630 nm as a reference for SAA. Lyophilized bovine acute phase serum was used as a standard; calibration was performed according to the European Union concerted action on standardization of animal APPs (No. QLK5-CT-1999-0153).

The livers were homogenized in phosphate buffered saline (PBS) and the homogenate was stirred in a vortex and centrifuged (2500 rpm) for 5 min.
The supernatant (10 μL) was added to each well in triplicate. PBS (0.2 mL), containing o-dianisidine dihydrochloride (4.2 mg), double-distilled water (22.5 mL), potassium phosphate buffer (2.5 mL, pH = 6), and H₂O₂ (10 μL, 1%) was also added. The enzyme reaction was stopped by addition of 30 μL sodium acetate (2.23 g in 20 mL of double-distilled water).
Myeloperoxidase activity was determined at 460 nm using a microplate spectrophotometer (Asys Expert Plus—Biochrom, Cambridge, UK).

The concentration of secretory immunoglobulin A (sIgA) in intestinal lavage was determined by a capture ELISA using Goat Anti-Mouse Ig, Human ads-UNLB (Southern Biotech) and Goat Anti-Mouse IgA (α chain specific) Horseradish Peroxidase (HRP) conjugate (Southern Biotech), as recommended by the manufacturer. The sample readings were performed using the Biochrom Asys Expert Plus microplate reader (Biochrom).

The concentrations of TNF-α, IL-6 and IL-10 in blood serum and uterine flush samples were determined using commercially available kits, i.e., bovine enzyme-linked imunosorbent assay (ELISA) kits for TNF-α, IL-6 and IL-10 (USCN Life Science Inc., Houston, USA). The inter- and intra-assay coefficients of variation (CV) for all examined cytokines were <12 and <10 %, respectively. All procedures were performed according to the guidelines provided by the manufacturers and methods in the literature (Kim et al. 2014 (link)). Absorbance readings were performed on an automatic microplate reader (Asys Expert Plus, Biochrom Ltd., Cambridge, England) at 450 nm.

IL-6 and IL-8 levels in the supernatants of cultures of an MLR and MSCs were determined using antibody-specific ELISA kits (PeproTech, Rocky Hill, USA) with internal controls according to the manufacturer’s instructions. The experiments were performed in triplicate and analyzed using an ELISA microplate reader at 450 nm Asys Expert Plus (Biochrom, Holliston, USA).

Gastrocnemius muscles of mice fed with HCD or HFD for 2 months were homogenized in phosphate-buffered saline (PBS), and the homogenate was stirred in a vortex and centrifuged (500 g, 4°C) for 5 min. The activity of MPO was measured in tissue supernatants (10 µL) in triplicate. PBS (0.2 mL) containing o-dianisidine dihydrochloride (4.2 mg), double-distilled water (22.5 mL), potassium phosphate buffer (2.5 mL, pH=6), and H₂O₂ (10 µL, 1%) was also added. The enzyme reaction was stopped by the addition of 30 µL sodium acetate (2.23 g in 20 mL of double-distilled water). MPO activity was determined at 460 nm, using a microplate spectrophotometer (Asys Expert Plus, Biochrom, UK), and is reported as absorbance (Ab).