Bio plex human cytokine 27 plex panel

In nAMD group, before the first intravitreal injection of anti-VEGF agents, approximately 0.1 mL of undiluted aqueous humor was collected by performing an anterior chamber limbal paracentesis. In control group, undiluted aqueous humor samples were obtained at the beginning of cataract surgery. The aqueous humor samples were transferred into sterile tubes and stored at −80 °C until processing. No complication associated with the sampling of aqueous humor occurred. Twenty-seven cytokines in the aqueous humor samples were measured by a Bio-Plex multiplex assay (Bio-Plex Human Cytokine 27-plex panel; Bio-Rad, Hercules, CA, USA) and a multiplex bead analysis system (Bio-Plex Suspension Array System; Bio-Rad) according to manufacturers’ instructions. All standards and samples were assayed in duplicate. Levels of aqueous humor cytokines below detectable levels were treated as 0 for statistical analysis^{9 (link),18 (link)}.

A Bio-Plex multiplex assay (Bio-Plex Human Cytokine 27-plex panel; Bio-Rad, Hercules, CA, USA) and a multiplex bead analysis system (Bio-Plex Suspension Array System; Bio-Rad) were used simultaneously to measure the levels of 27 types of cytokines in the aqueous humor, as previously described46 (link)47 (link)48 (link). The following were analyzed: IL-1b, 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 FGF, G-CSF, GM-CSF, IFN-γ, IP-10, MCP-1, MIP-1a, MIP-1b, PDGF-bb, RANTES, TNF-α, and VEGF. The levels of aqueous humor cytokines were set to 0 if the levels were below the detectable levels.

The culture supernatants of ARPE-19 cells incubated for 24 h were collected in sterile tubes and stored at −80 °C until assay. Twenty-seven types of cytokines in the culture supernatants were measured using the Bio-Plex Human Cytokine 27-Plex panel (Bio-Rad, Hercules, CA, USA) with a multiplex bead analysis system (Bio-Plex Suspension Array System; Bio-Rad) according to the manufacturer’s instructions. All standards and samples were assayed in duplicate. Cytokine levels below detectable levels were regarded as 0 μg/mL for statistical analysis [25 (link),26 (link)].
Some fluctuations of secreted cytokine levels occur, even if the experiments are strictly performed. In this study, preliminary experiments were conducted twice, and the results obtained in the third experiment were adopted in this study. The results in the preliminary experiments were almost the same as those in the third experiment.

The Bio-Plex ProTM magnetic color bead-based multiplex assay (Bio-Plex Human Cytokine 27-plex panel; Bio-Rad, Hercules, CA) was used to measure the concentrations of twenty-seven human aqueous humor cytokines: interleukin-1β (IL-1β), IL-1rα, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, basic fibroblast growth factor (b-FGF), EOTAXIN, granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GMCSF), interferon-gamma (IFN-γ), interferon-induced protein-10 (IP-10 or CXCL10), monocyte chemotactic protein-1 (MCP-1 or CCL2), macrophage inflammatory protein-1α (MIP-1α or CCL3), macrophage inflammatory protein-1β (MIP-1β or CCL4), platelet-derived growth factor-BB (PDGF-BB), regulated upon activation normal T-cell expressed and secreted (RANTES), tumor necrosis factor-alpha (TNF-α), and vascular endothelial growth factor (VEGF). The analysis procedure was performed according to the manufacturer's instructions. Standard curves were generated using the Bio-PlexTM 200 System (software version 6.0; Bio-Rad Laboratories) and were used to calculate the cytokine concentrations in the aqueous humor samples.

Plasma, PF, and urine samples were thawed on slush ice and analyzed using a multiplex cytokine assay (Bio-Plex Human Cytokine 27-Plex Panel, Bio-Rad Laboratories Inc., Hercules, CA) containing the following cytokines, chemokines and growth factors: interleukin (IL) 1 beta (IL-1β), IL-1 receptor antagonist (IL-1Ra), IL-2, IL-4, IL-5, IL-6, IL-7, IL-8 (CXCL8), IL-9, IL-10, IL-12 p70, IL-13, IL-15, IL-17, eotaxin (CCL11), basic fibroblast growth factor (bFGF), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), interferon gamma (IFN-γ), interferon gamma-induced protein/chemokine (C-X-C motif) ligand 10 (IP-10 or CXCL10), monocyte chemotactic protein 1 (MCP-1 or CCL2), macrophage inflammatory protein-1-alpha (MIP-1α or CCL3), macrophage inflammatory protein-1-beta (MIP-1β or CCL4), platelet-derived growth factor-BB (PDGF-BB), regulated upon activation T cell expressed and secreted (RANTES or CCL5), tumor necrosis factor (TNF), and vascular endothelial growth factor (VEGF). The analysis was performed according to the instructions from the manufacturer.

Plasma levels of IFABP, CCL25, IL‐18, IL‐18 binding protein (IL‐18BP) and LBP were measured in duplicate by enzyme immunoassays (EIA) using commercially available antibodies (R&D Systems, Minneapolis, MN, USA) in a 384 format using a combination of a SELMA (Jena, Germany) pipetting robot and a BioTek (Winooski, VT, USA) dispenser/washer. Absorption was read at 450 nm with wavelength correction set to 540 nm using an EIA plate reader (Bio‐Rad, Hercules, CA, USA). Plasma levels of IL‐1β and IL‐1Receptor antagonist (IL‐1Ra) were analysed using a multiplex cytokine assay (Bio‐Plex Human Cytokine 27‐Plex Panel; Bio‐Rad Laboratories Inc., Hercules, CA, USA). The samples were analysed on a Multiplex Analyzer (Bio‐Rad Laboratories) according to the manufacturer’s instructions.