Multiplex bead assay

Patients who agreed to blood draws underwent collection of venous blood at the preoperative visit (T0) within 2 weeks before surgery, as well as collection of venous blood on the mornings of postoperative day (POD) 1, POD 3, and POD5. Plasma samples were prepared and levels (pg/mL) were assessed using multiplex bead assay (Bio-Rad Laboratories, Hercules, CA, USA and EMD, Bioscience Research Reagents, Temecula, CA, USA) as previously described [65 (link)]. The 27 biomarkers (MIP-1b, IL-6, INFγ, IL-5, GM-CSF, TNFα, IL-2, IL-1b, IL-13, IL-4, MCP-1, IL-8, IL-10, G-CSF, VEGF, Resistin, IL-7, IL-12p70, IL-17, CXCL12, CXCL10, Omentin-1, Pentraxin-3, Galactin-3, TGFb1, TGFb2, and TGFb3) were selected by literature review for analysis given known variation with inflammatory stimuli and oncologic relevance in signaling pathways. The biomarkers sLeptin R, Vaspin, FGF-21, FGF-23, and PON-1 were included as portions of the multiplex bead assay. Acceptable signal quality to calculate the values (pg/mL) for these biomarkers was determined only by the values within the standards concentration range for each assay. To confirm reliability, all biomarker values were performed in duplicate on the established assay by a single research team member and all patient’s time point samples were arranged on to a single plate.

Blood was taken by a puncture of the saphenous vein, heart puncture, or retro-orbital plexus (Li-heparin-coated tubes; KADE, Nümbrecht, Germany) and centrifuged to separate cells and plasma. Total plasma IgE was measured in AROM+ and WT male mice at different age points using a classical immunoassay isotope-specific sandwich ELISA (BD Pharmingen). The determination of the immunoglobulin isotype levels (IgG1, IgG2a, IgG3, IgM and IgA) was performed either with a multiplex bead assay (Biorad, CA, USA) or with a combined electrochemiluminescence multiplexed assay system (Meso Scale Discovery, MSD, Rockville, MD USA).

Analyses were performed in February 2016, thus three months after enrolment of the final patient with NSTI and 11 months after the final control patient. Plasma levels of IL-1β, IL-6, IL-10 and TNF-α were determined using multiplex bead assays (Bio-Rad Laboratories, Hercules, CA, USA; Bio-Plex Pro^TM assay) according to the manufacturer’s instructions. Analyses were done using the Bio-Plex^® MAGPIX^TM Multiplex Reader (Bio-Rad Laboratories, Hercules, CA, USA). The samples were diluted in 1:4 and 1:10 against a standard pool with known concentration and the mean values were calculated after correction for the diluting steps. According to the manufacturer, the detection range without dilution was: IL-1β (0.2–556 pg/mL), IL-6 (5.2–18,618 pg/mL), IL-10 (1.1–11,850 pg/mL) and TNF-α (0.2–2,059 pg/mL) with an intra-assay variation of 8% and an inter-assay variation of 10%. Measurements below detection limit were set to 0. One patient had an IL-6 level above detection range, when taking the dilution factor into account, and was set to 180,000 pg/mL.

Cytokines and chemokines in BALF and serum samples from the mice were measured using multiplex bead assays (Bio-Rad, CA, United States) according to the manufacturer’s instructions. For analysis, the 96-well plate was placed in a Bio-Plex reader and the data collection, analysis and quality control were performed as previously described (Hye et al., 2014 (link)).