Luminex magpix analyzer

Mice metabolic hormones were determined using either specialized ELISA kit or Milliplex MAP Magnetic Bead Mouse Metabolic Hormone Panel (Millipore MMHMAG-44K, Merck, Germany). Plasma leptin and insulin were determined respectively by mouse leptin ELISA Kit (MultiSciences, China) and mouse insulin ELISA Kit (Beyotime, China) according to manufacturer’s instructions. The Milliplex panel was custom-designed for the simultaneous quantification of GIP (total), insulin, leptin and PYY (total). Plasma samples (10 μL) were assayed using a Luminex MAGPIX analyzer (Merck Millipore, Germany) and the data were processed by Milliplex Analyst 5.1 software and R v3.3.2 (R Core Team, 2016).

We used Luminex^® xMAP^® technology for analysis of proteins and cytokines in supernatants collected from primary fibroblasts or keratinocytes and cell cultures of ASCs stimulated with IM peptide at 0.1 µg/mL concentration. This method allows multiplex detection of proteins in single biological samples. For analysis of ASC supernatants we used Human Adipocyte Magnetic Bead Panel (Merck Millipore, Burlington, MA, USA) which enables simultaneous quantification of the following: Adiponectin, HGF, IL-1β, IL-6, IL-8, leptin, MCP-1, NGF, PAI-1 (total), resistin, and TNFα. For supernatants of fibroblasts, keratinocytes, and ASCs we assessed concentrations of 12 human cytokines and growth factors (angiopoietin-2, BMP-9, EGF, endoglin, endothelin-1, FGF-1 (acidic FGF), FGF-2 (basic FGF), follistatin, G-CSF, HB-EGF, HGF, IL-8, leptin, PLGF, VEGF-A, VEGF-C and VEGF-D) with Human Angiogenesis/Growth Factor Magnetic Bead Panel 1 (Merck Millipore, Burlington, MA, USA). The analysis was conducted according to the manufacturer’s instructions with a protocol described before by Deptula et al. [26 (link)]. Analysis was performed with a Luminex MAGPIX^® Analyzer (Merck Millipore, Burlington, MA, USA) and data was analyzed in xPONENT 4.2 software. Results are presented as the concentration of cytokines in units of pg per 1 mL.

Concentrations of VEGF-A, sEng, PlGF, and sFLT-1 were measured in thawed serum using the Luminex MAGPIX analyzer (Merck, Darmstadt, Germany) with kits purchased from R&D Biosystems (Minneapolis, MN; catalogue # LXSAHM; Lot # L127927) at our laboratory in Lusaka. VEGF-A was run separately from sEng, PlGF, and sFLT-1 to avoid antibody cross-reactivity. Samples were processed according to manufacturer instructions. Serum was diluted in calibrator diluent at a 1:3 ratio. A minimum of 50 microparticles were captured for each analyte.