Belysa immunoassay curve fitting software

Upon sacrifice, ~3 mL blood samples were collected via cardiac puncture from the left ventricle and placed in K₃ EDTA blood collection tubes. All blood samples were centrifuged at 5000 rpm for 10 min at 4 °C. Plasma was collected and immediately stored at −80 °C. Plasma cytokine and chemokine concentrations were measured according to manufacturer’s instructions, using a Luminex Magpix^® multiplex analyzer (27 Plex MILLIPLEX MAP Rat Cytokine/Chemokine Magnetic Bead Panel, Millipore Sigma, Burlington, MA, USA). Twenty-five microliters of undiluted plasma samples were run in duplicate, and the assay was analyzed using Belysa™ Immunoassay Curve Fitting Software (Millipore Sigma).
A separate set of female Sprague Dawley control rats of similar age were used to collect pre- and post-HBO plasma samples (n = 7–9/group). A catheter was placed in the tail vein prior to HBO exposure to collect pre-HBO blood samples. Animals were placed inside the HBO chamber, which was flushed with 100% O₂ and then pressurized to either 2 or 3 ATA. Immediately after the 1-h exposure, animals were removed from the chamber and blood was collected via the tail vein catheter. Plasma samples were prepared as described above and assayed via the 27 Plex MILLIPLEX MAP Rat Cytokine/Chemokine Magnetic Bead Panel.

At the end of experiments, all culture supernatants were harvested from each device (about 95 μl). Cells were removed by centrifugation at 1,500 rpm (about 1,000 g) for 10 min at 4°C, and 75 μl of each supernatant were collected and stored at −80°C until use. Soluble factor analyses were performed using a custom multiplex immunoassay panel for IL-6, IL-8/CXCL8, and GM-CSF (MILLIPLEX HCYTA-60K, MilliporeSigma). Samples were centrifuged again before use according to the manufacturer’s protocol. Assays were read on a Luminex 200 instrument (Luminex Corp.) with xPONENT software. Standard curves were obtained using Belysa^® Immunoassay Curve Fitting Software (MilliporeSigma). Data below the limit of quantification were plotted as zero on graphs.

Plasma cytokine and chemokine analyses at baseline and 24 h and 14 days after the first dose were performed using the ProcartaPlex NHP Cytokine & Chemokine Panel 30plex (Thermo Fisher Scientific) according to the manufacturer’s instructions. Samples were analyzed using a MagPix (Luminex) instrument, and the data were analyzed with Belysa Immunoassay Curve Fitting software (Millipore). Standard curves were generated using 5-parameter logistic (5-PL) curve fit.

Cytokine levels were determined using a multiplexed bead immunoassay. The cell supernatant sample was analyzed for IL-1β, IL-6, IL-10, TNF-α, and IFN-γ using a commercially available high-sensitivity human cytokine panel (Milliplex Map Kit; Millipore, Billerica, MA, USA) within a Luminex analyzer. Briefly, 25 µl of standards, quality controls and samples are added to the plate in duplicate. This was followed by 25 µl of magnetic beads. The plate was sealed, covered with foil, and incubated overnight on a plate shaker at 4 °C. The plate was washed three times and 25 µl of detection antibody was added to each well. After incubating the plate at room temperature (RT) for 1 h, 25 µl of streptavidin-phycoerythrin (PE) was added, per well. The plate was resealed and incubated for an additional 30 min at RT. The plate underwent a final series of washes before 150 µl of drive fluid was added. The concentrations of markers were measured on a Luminex® MAGPIX® instrument with Belysa® Immunoassay Curve Fitting Software (Millipore, Billerica, MA, USA). Quality control values for each marker were consistently within the range indicated by the manufacturer.

Serum supernatant fractions were thawed at room temperature and centrifuged at 1500 g for 10 min to remove remaining debris. Biomarkers in each sample were quantified using multiplex assays performed with MILLIPLEX® MAP magnetic bead panels according to the manufacturer's protocol (Table 2). All samples were run in duplicate, and plates were read on a Luminex® 100/200™ platform using xPonent® software (EMD Millipore Corp). Analyte concentrations were calculated using Belysa™ Immunoassay Curve Fitting Software (EMD Millipore Corp) and included in resulting analysis that provided >70% reliability.

Multiplex immunoassays (LXSAHM-03, R&D Systems, Mineapolis, MN, USA) were used to investigate fibroblast expression levels of interleukin-6 (IL-6), interleukin-1 receptor antagonist (IL-1RA), and platelet-derived growth factor alpha (PDGF-α) in cell lysates following the manufacturer’s instructions. The Luminex MAGPIX system with xPONENT 4.3 software (Luminex Corporation, Austin, TX, USA. A DiaSorin Company) instrument was used for analyses of the multiplex immunoassays (using Belysa ^® Immunoassay Curve Fitting Software (Merck KGaA, Darmstadt, Germany)). The ELISA assay (Human TGF beta 1 Elisa kit, ab108912, Abcam, Cambridge, UK) was further used for determining levels of transforming growth factor beta 1 (TGF-β1). Absorbance at 450 nm was measured using a 96 Plate Reader (Enzo Life Sciences, Inc., East Farmingdale, NY, USA) and additional background subtraction was done at 570 nm (analysis with Byonoy Software, Byonoy GmbH). Total protein quantification was done with a bicinchoninic acid assay (Sigma Aldrich, Merck KGaA, Darmstadt, Germany) and these data were used for normalization of the data.