Adipsin

Immune mediators in plasma were measured using a customized multiplex human factor panel (R & D Systems, Minneapolis MN) measuring cytokines (IFNβ, IFNγ, IL-1β, IL-10, IL-12p70, IL-13, IL-15, IL-17A, IL-18, IL-1RA, IL-2, IL-21, IL-4, IL-5, IL-7, TNFα, IL-23, IL-31, IL-22, IL-27), chemokines (CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1β, CCL5/RANTES, CCL11/Eotaxin, CXCL1/GROα, CXCL8/IL-8, CXCL9/MIG, CXCL10/IP-10, CXCL11/I-TAC, CXCL12/SDF-1α, CXCL13/BCA-1), growth factors (BDNF, GM-CSF, HGF, EGF, VEGF, PDGF-BB) and additional molecules (PD-L1, S100). Metabolic hormones were measured using a 3-plex kit measuring insulin, leptin, and PYY (Millipore, Burlington MA). Adipokines were assayed using a 5-plex kit measuring adiponectin, adipsin, lipocalin-2, total PAI-1, and resistin (Millipore, Burlington MA). CRP and IL-6 were measured in UCB plasma using a high-sensitivity ELISA (Life Technologies, Carlsbad CA) per the manufacturer’s instructions.
Supernatants from fetal rhesus macaque monocyte stimulation experiments were analyzed using an NHP XL Cytokine Premixed 36-plex kit (Bio-Techne, Minneapolis MN). Samples were diluted per the manufacturer’s instructions and analyzed in duplicate on the Magpix Instrument (Luminex, Austin, TX). Data were fit using a 5P-logistic regression on xPONENT software.

Cytokines, chemokines, and growth factors in undiluted cell culture supernatants and diluted UCB plasma (1:1) were measured using a human 37-plex luminex panel R & D Systems, Minneapolis MN). Metabolic hormones were measured using a 3-plex kit measuring insulin, leptin, and PYY (Millipore, Burlington MA). Adipokines were assayed using a 5-plex kit measuring adiponectin, adipsin, lipocalin-2, total PAI-1, and resistin (Millipore, Burlington MA). Samples were run in duplicates on the Magpix instrument (Luminex, Austin TX). Data were fit using a 5P-logistic regression on the xPONENT software. Values below the limit of detection were designated as half of the lowest limit. Data in pg/mL were tested for normality using Shapiro-Wilk test. Statistical differences in plasma proteins were tested using unpaired t-test with welch’s correction. Differences in protein in supernatants were tested using ordinary one-way ANOVA followed by Holm-Sidak’s multiple comparison tests. All statistical tests were performed on Prism 8 (GraphPad, San Diego, CA).

Immune mediators in plasma were measured using a customized multiplex human factor panel (R & D Systems, Minneapolis MN) measuring cytokines (IFNβ, IFNγ, IL-1β, IL-10, IL-12p70, IL-13, IL-15, IL-17A, IL-18, IL-1RA, IL-2, IL-21, IL-4, IL-5, IL-7, TNFα, IL-23, IL-31, IL-22, IL-27), chemokines (CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1β, CCL5/RANTES, CCL11/Eotaxin, CXCL1/GROα, CXCL8/IL-8, CXCL9/MIG CXCL10/IP-10, CXCL11/I-TAC, CXCL12/SDF-1α, CXCL13/BCA-1, growth factors (BDNF, GM-CSF, HGF, EGF, VEGF, PDGF-BB) and additional molecules (PD-L1, S100). Metabolic hormones were measured using a 3-plex kit measuring insulin, leptin, and PYY (Millipore, Burlington, MA). Adipokines were assayed using a 5-plex kit measuring adiponectin, adipsin, lipocalin-2, total PAI-1, and resistin (Millipore, Burlington, MA). Samples were diluted per the manufacturer’s instructions and run in duplicates on the Magpix Instrument (Luminex, Austin, TX). Data were fit using a 5P-logistic regression on xPONENT software. Group differences were compared using one-way ANOVA for unpaired samples followed by Holm-Sidak multiple test correction.