Kcjunior

A two-step immunoenzymatic method based on the ELISA test (Innovative Research Inc., Novi, MI USA) was used for the measurement of lysozyme levels [8 ]. The reading was performed at 405 nm, 30 min after adding the dye, using the µQuant reagent (Bio Tek, Winooski, VT, USA), while the processing of results was performed using the KCJunior (BioTek, Winooski, VT, USA). Human milk lysozyme was used as standard (Sigma-Aldrich). The obtained results were expressed in μg/mL. Imprecision of the method was 4.1%.

ADAM10 and ADAM17 protein concentrations in tissue homogenates were determined by enzyme immunoassay using ADAM10 and 17 assay kits (Cloud-Clone Corp., Houston, TX, USA) according to the manufacturer’s instructions. To determine the concentrations of the tested samples, a calibration curve was prepared using the standards included in the kit. Plates were read by Bio-Tek µQuant Universal Microplate Spectrophotometer (Bio-Tek, Winooski, VT, USA), using 450 nm as the primary wavelength. Data Analysis Software KCJunior (Bio-Tek, Winooski, VT, USA) was used. All standards and samples were run in duplicate. The absorbance was transformed into concentration.
The ADAM10 and 17 protein concentrations for each sample were normalized to the total amount of protein in the tissue lysates. Values are expressed in pg/µg protein for ADAM10 and for ADAM17 in ng/µg protein.
All immunoassays were performed at the Department of Medical and Molecular Biology, Faculty of Medical Sciences in Zabrze, Medical University of Silesia in Katowice.

The concentration of TNFα was measured in blood serum by ELISA method using goat anti-rabbit TNFα antibody as capture antibody and biotinylated, monoclonal anti-rabbit TNFα antibody as tracer (both from BD Pharmingen, USA). The assay was performed according to the manufacturer's instruction: goat antibody was immobilized on ELISA plates (Maxisorp, Nunc, Denmark) and bovine serum albumin was used for blocking of unbound sites. Standard curve was constructed with the use of rabbit TNFα (0,05–10 ng/mL; BD Pharmingen, USA) in BSA solution. Serum samples and TNFα standards were incubated for 2 hours and washed out with PBST. Next, biotinylated anti-TNFα was incubated for 1 hour. Immobilized immunocomplexes were detected with streptavidin-horseradish peroxidase conjugate (Dako-Cytomation, Denmark; 30 minutes) and visualized using TMB Supersensitive System (Sigma-Aldrich, USA). Then, the reaction was stopped with 0,5 M sulfuric acid. The absorbance was measured on PowerWave XS ELISA plate reader (BioTek, USA; 450 nm/630 nm as reference). KCJunior (a computer program) (BioTek, USA) was used to collect data. Results were presented as pg of TNFα per mL of serum [pg/mL]. Interassay error was 6.4%.

The commercial ELISA (Diapra, Italy) was used to determine cortisol and testosterone levels. The analytical procedure was in accordance with the manufacturer's instructions in the technical manuals supplied with the kits. Absorbance readings were taken using a µQuant reader (Biotek, USA), while the results were processed using KCJunior (Biotek, USA). The method’s sensitivity was 0.12 ng/mL for cortisol and 3.28 pg/mL for testosterone. The method's imprecision was 6.2% and 7.9%, respectively.
Total protein was determined using the Lowry method: This method uses the reactions between peptide bonds and tyrosine and Folin-Ciocalteu reagent. The absorbance of the resulting color was read at 650–750 nm, 30 min after the reagent addition. Bovine serum albumin water solution (BSA – Sigma Aldrich, Germany) at slightly basic pH was used as standard. The results obtained were presented in mg/mL. Measurement imprecision of the method was 6.5%.

Cell viability assays were carried out with Cell Counting Kit-8 (RiboBio, China) according to the manufacturer’s instructions. In brief, 10 μL CCK8 solution in 100 μL complete culture medium was added to each well of the 96-well plate and further incubated for 3 h. Then, the absorbance was measured at 450 nm optical density in a microplate reader (KC junior, BioTek, United States). The cell viability was calculated by the mean of the optical density values in 6 repeat wells.