Magpix multiplex reader

Venous blood was collected from healthy controls and BA patients at the time of KPE into a sterile ethylenediamine tetraacetic acid (EDTA)-containing tube. Plasma samples were separated by centrifugation at 1,500 g for 10 min and subsequently stored at −80°C for subsequent analysis. Systemic concentrations of cytokines in BA patients and healthy controls were measured using the Bio-Plex Pro Human Cytokine 27-Plex Assay on the Bio-Rad MAGPIX Multiplex Reader (Bio-Rad, Hercules, CA, USA) following the manufacturer’s instructions. The analyzed cytokines were as follows: (1) inflammatory cytokines including IL-1β, IL-6, IL-7, IL-8, IL-9, and TNF-α; (2) immunomodulatory cytokines including IL-2, IL-12p70, IL-15, IL-17, and IFN-γ; (3) chemokines including eotaxin, IFN-γ-induced protein 10 (IP-10), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein (MIP)-1α, MIP-1β, and RANTES (Regulated on Activation, Normal T Expressed and Secreted, CCL5); (4) growth factors including granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF); and (5) anti-inflammatory cytokines including IL-1 receptor antagonist (IL-1ra), IL-4, IL-5, IL-10, and IL-13.

Serum samples (SST; 5 ml) were left for at least 30 min to clot at room temperature, before being centrifuged at 1300×g for 15 min. Plasma samples (EDTA; 6 ml) were spun immediately after collection at 1300 g for 15 min. The supernatant was then aliquoted in triplicate and stored at − 80 °C for later analysis. Samples were batch analysed for plasma glucose using a commercial kit (GAHK20, Sigma-Aldrich, MO, USA); serum APN using a multiplex cytokine assay (Bio-Plex Pro Human Diabetes Assay); MMP2, MMP-3 and OPN (Isoform B) (Bio-Plex, 37-plex Human Inflammation Panel) using the MAGPIX multiplex reader (Bio-Rad, Richmond, CA, USA).

Forty female ovariectomized albino C57BL/6 mice were singly housed and randomized to be fed either a DIO diet (60% kcal from fat; Research Diets # D12492) or a low fat, isonutrient matched control (10% kcal from fat; Research Diets # D12450J) ad libitum for 13 weeks before being harvested for body composition and fasting serum hormone analysis (4 h fasting, n = 5/group). Body fat and lean mass was measured using a Lunar Piximus X-Ray imager (GE Medical Systems, Ontario, CA). Serum hormones leptin, resistin and insulin were measured using a mouse diabetes multiplex assay on a MAGPIX multiplex reader (BioRad, Hercules, CA). The remaining mice received a tail vein injection of metM-Wnt^lung cells (2.5 × 10³/mouse in 200 μl; n = 15/group, G*Power analysis) and were maintained on their diet regimens. Mice were monitored carefully and were harvested upon signs of illness. Two mice from the DIO group were excluded upon being found dead in their cage. Incidence of macrometastases and micrometastases was determined by histological analysis and was validated by the Animal Histopathology Core at University of North Carolina at Chapel Hill.

Serum cytokine and chemokine concentrations (pg/ml) were measured using a Bio-Rad Mouse 23-plex kit (Bio-Rad, Hercules, CA, USA). Calibration curves from recombinant cytokine and chemokine standards were prepared for the 8-point standard dilution set with 4-fold dilution steps in sterile PBS. The samples were measured using a Bio-Plex MagPix Multiplex Reader (Bio-Rad Laboratories Inc. by the Luminex Corporation, The Netherlands). The Bio-Plex Manager software's five-parameter logistic curve fitting (5PL) method was used for raw data analysis and calculation of cytokine concentrations.

Human blood samples were incubated with PBS, MPLA, or LPS at 37 °C for 6 hours and 24 hours, and then the supernatant was harvested for cytokine production measurement. Interferon beta-1 (IFNB1) (41415, PBL Assay Science), IL-23 (88-7237 eBioscience), and CCL7 (88-50700, eBioscience) concentrations were measured using ELISA according to the manufacturer’s protocols. Cytokine concentrations were determined by measuring optical density at 450 nm using a microtiter plate reader (Dynatech Laboratories, Chantilly, VA, USA). Concentrations of IL-6, IL-1α, TNF-α, IL-8, IL-12B, IFN-γ, CXCL10, IL-1β, and IL-18 were measured by use of a customized Bio-Plex Multiplex Assay and MAGPIX Multiplex Reader (Bio-Rad Laboratories). Results were analyzed with Bio-Plex Manager Software 6.1, and graphs were made with GraphPad Prism Software 6.0 (GraphPad Software, San Diego, CA, USA).