Bio plex pro human cytokine 27 plex

Before measuring 27 cytokines, vitreous samples were centrifuged 10,000g for 10 min to remove cellular components. Vitreous samples were measured using a Bio-Plex Pro Human Cytokine 27-plex (Bio-Rad Cat# M500KCAF0Y, RRID:AB_2893118, California, USA), which included the following 27 cytokines: interleukin (IL)-1β, IL-1 receptor antagonist (IL-1ra), IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, Eotaxin, basic fibroblast growth factor (b-FGF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-γ (IFN-γ), interferon-inducible 10 kDa protein (IP-10), monocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein (MIP)-1α, MIP-1β, platelet-derived growth factor (PDGF), regulated upon activation, normal T cell expressed and secreted (RANTES), tumor necrosis factor alpha (TNF-α), and VEGF. The experimental procedure for the cytokine assay was in accordance with the instructions. When the measured values fell below the limit of detection, we set the recorded concentration at the limit of detection. A cytokine was excluded if more than 10% of the samples were below the detection limit.

Phosphate-buffered saline, Roswell Park Memorial Institute 1640 (RPMI-1640) medium, penicillin–streptomycin 100×, IL-2, and LPS were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). CD69 fluorescein isothiocyanate, CD56 phycoerythrin, CD3 peridinin chlorophyll protein, CD25 brilliant violet 421 and heparin Vacutainer vials were purchased from BD Biosciences (Franklin Lakes, NJ, USA). The Bio-Plex Pro™ human cytokine 27-Plex was purchased from Bio-Rad Laboratories Inc. (Hercules, CA, USA).