Bio plex human cytokine assay

Cytokine levels come from those reported in Nicholson et al. [16 ] and were measured as described by use of a Bio-Plex Human Cytokine Assay (Bio-Rad Laboratories) including interleukin (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, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon (IFN)-γ, monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1a, MIP-1b, vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF)-bb, basic fibroblast growth factor (bFGF), chemokine (C-C motif) ligand (CCL) 2, CCL3, CCL4, CCL5, C-X-C motif chemokine 10 (also known as IP-10), eotaxin and tumor necrosis factor (TNF)-α.
Caspase and LDH levels were measured as described [15 (link)] using the Caspase-Glo 3/7 Assay (Promega, Madison, WI, USA) and Cytotoxicity Detection Kit (Roche Applied Science, Indianapolis, IN, USA), respectively [15 (link)]. MMP-7 and MPO levels were measured using Human Total MMP-7 and Human Myeloperoxidase Quantikine ELISA Kits (R&D systems Minneapolis, MN, USA).

A multiplex biometric immunoassay, containing fluorescent microspheres conjugated with a monoclonal antibody specific for a target protein, was used for cytokine measurement according to the manufacturer's instructions (Bio-Plex Human Cytokine Assay # M50–0KCAF0Y; Bio-Rad Inc., Hercules, CA, USA). For this assay, six patients from the control group had plasma available. All seven plasma samples from T1DM patients were tested in this assay.

Anti-inflammatory activity of VLAM and VCAM was evaluated by inhibition of tumor necrosis factor α (TNF-α) release from activated human peripheral blood mononuclear cells (PBMCs). A vial of cryopreserved PBMCs (Precision for Medicine, Bethesda, MD) was thawed, and cells were resuspended in DMEM+10% FBS at a concentration of 1x10⁶ cells/ml. PBMCs were stimulated with Cluster of Differentiation (CD) CD3/CD28 antibodies (BD Biosciences, San Jose, CA) at 10 ng/ml and incubated for 48 h in the presence of a 12 cm² of VCAM or VLAM, which were cut with a scalpel into 6–8 smaller pieces prior to placing in a 24-well plate at 37° C and 5% CO₂. Stimulated and unstimulated PBMCs alone served as positive and negative controls, respectively. After 48 h, culture supernatants from each well were collected and centrifuged at 14,000 rpm for 5 min. TNF-α levels in each supernatant were quantified using a Bioplex human cytokine assay (BioRad, Hercules, CA) according to the manufacturer’s protocol.

The levels of 16 cytokines (IL-1β, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IFN-γ, MIF, TNF-α, and TNF-β), 8 chemokines (CCL2, CCL3, CCL4, CCL5, CCL11, CXCL10, CXCL12 and IL-8), 7 growth factors (FGF-2, G-CSF, GM-CSF, HGF, IL-7, PDGF and VEGF) and 2 adhesion molecules (ICAM-1 and VCAM-1) in dengue patients’ sera were evaluated via BioPlex Pro Assays (Bio-Plex Human Cytokine Assay; Bio-Rad Inc., Hercules, CA, USA) [19] (link). The fluorescent signals were read using the Bio-Plex 200 System (Bio-Rad Laboratories). Via the Bio-Plex Manager 6.0 Software, raw data was measured as the relative fluorescence intensity and then converted to cytokines concentration based on the standard curve generated (Bio-Rad Laboratories). Once again, the ND and DWoWS patients were grouped as “without warning signs” and the DwWS and SD patients were grouped as “with warning signs”. The differences in cytokine levels between the differences groups dengue patients were evaluated using the Kruskal-Wallis one way analysis of variance (ANOVA), followed by Dunn’s multiple comparison test. All statistical analyses performed were done using GraphPad Prism 5 for Windows, Version 5.01 (San Diego, California, USA).

Following euthanasia, bronchoalveolar lavage fluid (BALF) was collected for subsequent ELISA tests. The samples were then centrifuged at 5000 rpm, 4 °C for 15 min, and the supernatant was collected and stored at −80 °C for cytokine measurement. The levels of TNF-α, IL-1β, IL-6, GM-CSF, and TSLP in the BALF were quantified using enzyme-linked immunosorbent assays (ELISA) kits purchased from R&D Systems (Minneapolis, MN, USA). ELISA was performed following the manufacturer’s instructions, and values were expressed as pg/mL. For in vitro assays, supernatant samples from macrophages were processed using a multiplex biometric immunoassay. Monoclonal antibodies specific for the target proteins were used to measure cytokines MCP-1, MIP-1, and RANTES according to the manufacturer’s instructions (Bio-Plex Human Cytokine Assay; Bio-Rad Inc., Hercules, CA, USA).

Multiplex biometric immunoassay containing fluorescent dyed magnetic beads conjugated with monoclonal antibodies specific for the targeted 10 protein biomarkers was used according to the manufacturer's instructions (Bio-Plex Human Cytokine Assay; Bio-Rad Inc., Hercules, CA, USA) [23 (link)]. Following protein extraction and purification, GCF samples were diluted to a ratio of 1:4 and incubated with the magnetic beads. After a series of washes with Bio-Plex Pro wash station, biotinylated detection antibody was added to create a sandwich complex. Thereafter, Streptavidin-Phycoerythrin conjugate was added as a fluorescent indicator. A range of (352290–0.97) pg/ml recombinant cytokines was used to establish the standard curves. Biomarkers quantities were determined using a multiplex array reader powered by Luminex-200 System. The amounts were calculated using Bio-Plex Manager software and reported as picograms per 30 seconds (pg/30 s) [24 (link)].