Q analyzer tool

Mouse sera of the ORFV-treated and PBS-treated subjects were collected and measured, according to the manufacturer's instructions, with a semi-quantitative mouse cytokine antibody array (RayBio Human Cytokine Antibody Array G series 4000; RayBiotech, Inc.) which detects 200 cytokines in one experiment. Briefly, the samples were added to the cytokine antibody chips, then incubated with the biotinylated antibody. Cy3 Equivalent Dye-Streptavidin was added, and the fluorescence signals were visualized through a laser scanner equipped with a Cy3 wavelength (InnoScan 300 Microarray Scanner; Innopsys). The mean fluorescent signals of each group containing three repeats were analyzed with specific software Q-Analyzer Tool (code: QAM-CAA-4000) provided by RayBiotech, Inc. The cytokines (FC ≥2 or FC ≤0.5; Table I) were noted by functional category, comprehensively referring to the COSMIC database (http://cancer.sanger.ac.uk/cosmic/), GeneCards database (http://www.genecards.org/), NCBI Gene database (https://www.ncbi.nlm.nih.gov/gene/), and references (25 (link)–31 (link)).

Crosslinked AMs and normal AMs were placed in 6-well plates; 1 ml of serum-free Gibco Dulbecco's modified Eagle medium (DMEM; Thermo Fisher Scientific) was then added to each well. Each piece was 9 cm² in size. The supernatants incubated for 24 hours in serum-free medium were obtained to determine the protein content of the supernatants and the growth factors present in each sample. The analysis of growth factor levels was carried out using a Quantibody Human Growth Factor Array kit (RayBiotech, Peachtree Corners, GA, USA), following the manufacturer's protocols. Briefly, after a 30-minute incubation at room temperature with sample diluent, the glass chips were washed five times, and each well arrayed with human growth factor antibodies was overlaid with 100 µl of medium. After overnight incubation at 4°C and extensive washing, the detector antibody was added for 1 hour and then washed away, and Alexa Fluor 488 conjugated streptavidin was added for 2 hours at room temperature. The signals were scanned with a fluorescence laser scanner. Each sample was prepared in quadruplicate. Growth factors were quantified with the Q-Analyzer tool (#QAH-GF-1-SW; RayBiotech) against a standard curve set for each growth factor, with a five-point serial dilution of growth factor standards.