The Bio-Plex Pro human chemokine panel (40-plex, Bio–Rad Laboratories, Hercules, CA) and the Bio-Plex Pro human inflammation 1 panel (37-plex, Bio–Rad Laboratories) were used for the cytokine/chemokine measurements in the sera of patients as previously reported. There were several duplicates of cytokines in the two assay kits and finally 73 cytokines (S1 Table ) were measured. Both assay kits contained heterophilic antibody blocking reagents to inhibit rheumatoid factor interference in the measurements.
Bio plex pro human inflammation panel 1
Manufactured by Bio-Rad
Sourced in United States
The Bio-Plex Pro Human Inflammation Panel 1 is a multiplex assay that allows for the simultaneous measurement of multiple inflammation-related analytes in a single sample. The panel is designed to provide a comprehensive assessment of the inflammatory state in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Bio plex pro human chemokine panel, by Bio-Rad (2 mentions)
Lipofectamine messengermax, by Thermo Fisher Scientific (2 mentions)
Poly d lysine coated cell culture plate, by Greiner (2 mentions)
Bio plex pro 200 system, by Bio-Rad (2 mentions)
Bio plex manager software, by Bio-Rad (1 mentions)
Fungitell kit, by Associates of Cape Cod (1 mentions)
Luminex high performance assays, by R&D Systems (1 mentions)
Quantikine kits, by R&D Systems (1 mentions)
Duoset elisa kit, by R&D Systems (1 mentions)
Flexmap 3d reader, by Bio-Rad (1 mentions)
Versamax, by Molecular Devices (1 mentions)
Luminex xmap, by Bio-Rad (1 mentions)
11 protocols using bio plex pro human inflammation panel 1
1
Cytokine and Chemokine Profiling of Patient Sera
2
Multiplex Serum Immunoassay Analysis
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All serum samples were kept frozen at − 80 °C until use. Assays were performed according to manufacturer’s instructions using the Bio-Plex Pro human chemokine panel (40-plex, Bio-Rad Laboratories, Hercules, CA), and the Bio-Plex Pro human inflammation 1 panel (37-plex, Bio-Rad Laboratories). Both assay kits include heterophilic antibody blocking reagents to inhibit the rheumatoid factor interference in the measurements. Bio-Plex Manager software (ver. 6.1, Bio-Rad Laboratories) was used to fit the calibration curve data using a five-parameter logistic regression model where recovery was in the 70–130% range, and to determine the observed concentrations of analytes in serum samples. For measured values out of range, the values above the upper and lower limits of quantification were replaced by the highest or lowest value available, respectively.
3
Phage-Induced Airway Cytokine Profiling
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Phages associated with the airway epithelium were determined by treating CFBE41o- cells grown in Transwell inserts with phages OMKO1, LPS-5, PSA04, and PSA34 at a concentration of 1 × 109 PFU/ml in MEM pH-adjusted to 6.5 at 37°C for 24 h. Apical and basolateral media was collected, centrifuged at 370 × g at 4°C for 5 min, and human proinflammatory cytokines were quantified using the Bio-Plex Pro Human Inflammation Panel 1 (BioRad). Fluorescence was read using a MAGPIX System (Luminex) at the UPMC Children’s Hospital of Pittsburgh (Pittsburgh, Pennsylvania, USA). Cytokines that showed values below the limit of detection were discarded from the analysis.
4
Biomarkers of Inflammation and Intestinal Damage
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Biomarkers were measured on EDTA plasma or serum. Intestinal fatty acid binding protein (I-FABP) was measured using the DuoSet ELISA Kit; sCD14 and IL-6 were measured by ELISA using Quantikine kits; sCD163 and sTNFRII were measured using Luminex high-performance assays (all R&D systems, Minneapolis MN). Serum (1,3)-β-D-glucan was measured using the Fungitell® kit (Associates of Cape Cod, Inc., Falmouth, MA). Other inflammation biomarkers were measured using the Luminex platform (Bio-Plex Pro™ Human Inflammation Panel 1, Bio-Rad Laboratories, Inc., Hercules, CA).
5
IFN-β Secretion Assay in BJ Fibroblasts
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BJ fibroblasts were seeded in 96-well poly-d -lysine-coated cell culture plates (Greiner) at 20 000 cells per well and transfected the next day with 50 ng per well of the respective mRNA using Lipofectamine™ MessengerMAX™ (Life Technologies). The supernatant was harvested 48 h post-transfection and interferon-β (IFN-β) levels were determined using the Bio-Plex Pro Human Inflammation Panel 1 (BioRad) as per the manufacturer's protocol. Data were acquired on the Bio-Plex Pro 200 system (BioRad).
6
Measuring Soluble Biomarkers in CSF and Plasma
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Levels of soluble biomarkers were measured in CSF and plasma via a combination of Luminex-based custom assays and ELISAs, as per manufacturers’ protocol and as described previously [31 (link)]. IP-10/CXCL10 and IL-10 were measured using the Luminex based Milliplex Map Human Cytokine / Chemokine Panel (Millipore Sigma, St. Louis MO). Soluble CD163 was measured via the Bio-Plex Pro Human Inflammation Panel 1 (Bio-Rad Laboratories, Hercules CA). Neopterin was measured by chemiluminescent detection ELISA (Genway Biotech, San Diego CA). Data was collected on a FlexMap 3D reader (Bio-Rad Laboratories, Hercules CA) or VersaMax plate reader (Molecular Devices, Sunnyvale CA) and analyzed in Prism v.9 for Mac OS X (GraphPad, La Jolla CA) using 4-parameter fit standard curves.
7
IFN-β Secretion Assay in BJ Fibroblasts
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BJ fibroblasts were seeded in 96-well poly-d -lysine-coated cell culture plates (Greiner) at 20 000 cells per well and transfected the next day with 50 ng per well of the respective mRNA using Lipofectamine™ MessengerMAX™ (Life Technologies). The supernatant was harvested 48 h post-transfection and interferon-β (IFN-β) levels were determined using the Bio-Plex Pro Human Inflammation Panel 1 (BioRad) as per the manufacturer's protocol. Data were acquired on the Bio-Plex Pro 200 system (BioRad).
8
Multiplexed Cytokine Quantification from Serum
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This robust immunoassay facilitates the quantification of multiple protein biomarkers from a single sample in the single well of a 96-well plate in only 3–4 h using as little as 12.5 µL of serum, as per the manufacturer’s instructions (Bio-Plex Pro Human Inflammation Panel 1 (Bio-Rad, USA)). A magnetic bead assay was performed to analyze the levels of different cytokines/chemokines (IFN-α2, IFN-β, IFN-γ, IL-2, IL-8, IL-10, IL-12p40, IL-12p70, IL-26, IL-27 (p28) and IL-35). The concentration of each analyte bound to each specific set of beads was calculated based on a standard curve and is presented in pg/mL. Data from the reactions were acquired using a Bio-Plex three-dimensional (3D) suspension array system. Bio-Plex Data Software (xPPONENT software v.4.2.1441.1) was used to quickly visualize the results and for simple statistical analysis. The lower and upper limits of quantification (LLOQ and ULOQ) were inputted from the standard curves included in the kit.
9
Multiplexed Immunoassay for IL-8
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Tissue concentrations of IL-8 were detected by a bead-based multiplexed immunoassay system (Luminex xMAP, Bio-Rad, Hercules, CA, USA) with the commercial kits (Bio-Plex Pro Human Inflammation Panel 1, Bio-rad, Hercules, CA, USA) according to the manufacturer’s instructions. The limit of detection of the assay was 2.7 pg/ml for IL-8.
10
Phage Mediated Airway Inflammation
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Phages associated with the airway epithelium were determined by treating CFBE41o- cells grown in Transwell inserts with phages OMKO1, LPS-5, PSA04, and PSA34 at a concentration of 1 x 109 PFU/ml in MEM pH-adjusted to 6.5 at 37°C for 24 h. Apical and basolateral media was collected, centrifuged at 370 x g at 4°C for 5 min, and human proinflammatory cytokines were quantified using the Bio-Plex Pro™ Human Inflammation Panel 1 (BioRad). Fluorescence was read using a MAGPIX System (Luminex) at the UPMC Children’s Hospital of Pittsburgh (Pittsburgh, PA). Cytokines that showed values below the limit of detection were discarded from the analysis.
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