Manager software v 6

The levels of 8 selected cytokines (Interferon gamma (IFN-g), Monocyte chemoattractant protein-1 (MCP-1) (CCL2), Macrophage inflammatory protein (MIP)-1b, Interleukin (IL)-1b, Matrix metallopeptidase (MMP)-9, Interleukin (IL)-6, Interleukin (IL)-8 and regulated and normal T cell expressed and secreted (RANTES) (CCL5)) were measured in the WBA and LPA supernatants using an 8-plex analyte-specific antibody kit (Luminex Human Magnetic Assay, R&D Systems). All samples were diluted 1:2 and the assay were done according to the Manufacturer’s instructions. The assays were performed and read in a central laboratory on the Bio-Plex platform (Bio-Plex® MAGPIX™ Multiplex reader (MAGPIX13046704, BioRad Laboratories, Inc., California, USA).), with the Bio-Plex Manager Software V.6.1 used for bead acquisition and analysis.

Cell supernatants derived from HUVECs in normoxia and anoxia were collected at 8 h (cells used for RNA Seq), spun down at 1000× g for 10 min at 4 °C to remove any cell debris, and stored after addition of protease inhibitors (cComplete Mini; Roche, Basel, Switzerland) at −80 °C until analyses. A Bio-Plex Assay plate (Bio-Plex Pro Hu Screening Panel 27plx XPL) was purchased from Bio-Rad (Hercules, CA, USA) to detect human cytokines, interleukins, and other mediators and used according to the manufacturer’s recommendations. This kit permitted the detection of the following cytokines: FGF, G-CSF, IL-1β, IL-1α, IL-3, IL-16, IL-2, IL-5, IL-6, IL-8, LIF, IL-12, IL-15, MCP-1, SCF, SCGF-β, PDGF-BB, VEGF, MIF, M-CSF. Plates were read using a BioPlex^® 200 instrument (Bio-Rad, Hercules, CA, USA), and interleukin concentrations (expressed as picograms per milliliter) were calculated by use of a standard curve and software provided by the manufacturer (Bio-Plex manager software, v.6.1) and normalized by protein content defined by the Bradford assay.