Quantification of IFN-α, IFN-β, IFN-λ1, TNF-α, IL-6, IL-1β, and IP-10 secreted by human DCs after 12 h and by NHBE cells after 48 h of infection with different NS1-expressing recombinant viruses was performed with a multiplex ELISA kit (ProcartaPlex Assay; Invitrogen) according to the manufacturer’s recommendations. Data were analyzed by xPONENT software for the Luminex MAGPIx System.
Procartaplex assay
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ProcartaPlex assay is a multiplex immunoassay platform developed by Thermo Fisher Scientific. It enables the simultaneous measurement of multiple analytes in a single sample, allowing for efficient and comprehensive analysis of biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Luminex 200 instrument, by Thermo Fisher Scientific (2 mentions)
Human il 22 quantikine elisa kit, by R&D Systems (2 mentions)
Smctm human il 22 high sensitivity immunoassay kit, by Merck Group (2 mentions)
Smc pro, by Merck Group (2 mentions)
Elisa maxtm deluxe set mouse il 22, by BioLegend (2 mentions)
Human s100a8 duoset elisa, by R&D Systems (2 mentions)
Magpix system, by DiaSorin (1 mentions)
Luminex 200 machine, by DiaSorin (1 mentions)
Procartaplex panel 1a, by Thermo Fisher Scientific (1 mentions)
Non enzymatic cell dissociation buffer, by Merck Group (1 mentions)
Procartaplex, by Thermo Fisher Scientific (1 mentions)
Duoset elisa development system, by R&D Systems (1 mentions)
Complete edta free, by Roche (1 mentions)
Zombie nir, by BioLegend (1 mentions)
Facsaria 3 cell sorter, by BD (1 mentions)
Luminex 200 system, by Thermo Fisher Scientific (1 mentions)
Bio plex 200 system, by Bio-Rad (1 mentions)
Luminex 200 instrument, by DiaSorin (1 mentions)
Procartaplex simplex kit, by Thermo Fisher Scientific (1 mentions)
16 protocols using procartaplex assay
1
Quantification of Antiviral Cytokines
2
Cytokine Profiling in Colon and Serum Samples
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To prepare protein lysates from colon tissue, 1 cm of the proximal colon was harvested, washed with PBS, and minced into small pieces (∼1 mm). Colon pieces were resuspended in 500 μl nonenzymatic cell dissociation buffer (Sigma-Aldrich) and treated by sonication on ice for 1 min for 20 timed cycles using the Ultrasonic Processor W-385, Heat Systems-Ultrasonics, Inc. Cycle time was set to 2 s, 60% duty cycle at maximum output control. Samples were centrifuged at 2,000 g for 10 min at 4°C to remove cell debris. Supernatants were collected and stored at −80°C until analysis.
For serum preparation, whole blood was collected by cardiopuncture and allowed to clot undisturbed at room temperature for 30 min. Clots were removed by centrifugation at 2,000 g for 10 min at 4°C. Serum samples were collected and stored at −80°C until analysis.
Cytokines in colon lysates and serum were measured by the ProcartaPlex assay (mouse ProcartaPlex panel 1A; Invitrogen) performed on a Luminex 200 machine (Luminex). Cytokines and chemokines analyzed were as follows: CXCL1 (C-X-C motif chemokine 1), CXCL2, CXCL5, CXCL10, CCL2 (C-C motif ligand 2), CCL3, CCL4, CCL5, CCL7, Eotaxin, IFN-α, IFN-γ, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-13, IL-15/IL-15R, IL-17A, IL-18, IL-22, IL-23, IL-27, IL-28, IL-31, G-CSF, GM-CSF, M-CSF, leukemia inhibitory factor, and TNF-α.
For serum preparation, whole blood was collected by cardiopuncture and allowed to clot undisturbed at room temperature for 30 min. Clots were removed by centrifugation at 2,000 g for 10 min at 4°C. Serum samples were collected and stored at −80°C until analysis.
Cytokines in colon lysates and serum were measured by the ProcartaPlex assay (mouse ProcartaPlex panel 1A; Invitrogen) performed on a Luminex 200 machine (Luminex). Cytokines and chemokines analyzed were as follows: CXCL1 (C-X-C motif chemokine 1), CXCL2, CXCL5, CXCL10, CCL2 (C-C motif ligand 2), CCL3, CCL4, CCL5, CCL7, Eotaxin, IFN-α, IFN-γ, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-13, IL-15/IL-15R, IL-17A, IL-18, IL-22, IL-23, IL-27, IL-28, IL-31, G-CSF, GM-CSF, M-CSF, leukemia inhibitory factor, and TNF-α.
3
Multiplexed Cytokine and Chemokine Profiling
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Multiple cytokines and chemokines (IFN-α, IFN-γ, IL-6, IL-10, IL-17A, monocyte chemoattractant protein 1 [MCP-1], RANTES, and TNF-α) were measured using a custom 8-plex panel mouse ProcartaPlex assay (ThermoFisher Scientific; catalog no. PPX-08-MXGZGFX), following the manufacturer’s instructions. The assay was performed in a BSL3 laboratory, and samples were decontaminated by an overnight incubation in 10% formaldehyde solution before readout on a Luminex 100/200 system running on Xponent v4.2 with the following parameters: gate 5,000 to 25,000, 50 μL of sample volume, 50 events per bead, sample timeout 120 s, and low photomultiplier tube (PMT) (LMX100/200: Default). Acquired data were analyzed using ProcartaPlex analysis software v1.0.
4
Intracellular Cytokine Profiling in Virus-Infected Cells
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Cells were seeded at a density of 1 × 105 cells/cm2 and infected with Tha or Th2P-4M at an MOI of 5. Forty-eight hours post-infection, cells were lysed using Procartaplex™ cell lysis buffer (EPX-99999-000, Thermo Scientific) according to the manufacturer’s instructions to quantify protein expression in the cellular cytoplasm. Intracellular protein concentrations of human IFN-β, IFN-γ, IL-1β, IL-6, IL-15, CXCL10, LIF, CCL5, and TNF-α were quantified using a 9-plex Procartaplex assay (PPX-09, Thermo Scientific). In short, DropArray 96-well-plates (96-CC-BD-05, Clinisciences, Nanterre, France) were blocked using 1% BSA for 30 min. After blocking, 20 µL of cell lysate was added per well. According to the protocol, plates were stepwise incubated with 5 µL detection antibody, 10 µL streptavidin-PE, and 10 µL reading buffer per well before being read by the Luminex 200™ instrument (Thermo Scientific).
5
Multiplex Cytokine Quantification in Mouse
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Multiple cytokines and chemokines (IFN-α, IFN-γ, IL-10, IL-17A, IL-6, MCP-1 or CCL2, RANTES or CCL5, TNF-α) were measured using a custom 8-plex panel mouse ProcartaPlex assay (ThermoFisher Scientific; catalog no. PPX-08-MXGZGFX, lot 279751-000) by following the manufacturer’s instructions. An immunoassay was performed in the ABSL3 laboratory, and samples were decontaminated by an overnight incubation in 1% formaldehyde solution before readout on a Luminex 100/200 system running on xPONENT v4.2 with the following parameters: gate, 5,000 to 25,000; 50 μl of sample volume; 50 to 100 events per bead; sample timeout, 120 s; and low photomultiplier tube (PMT) (LMX100/200 default). Acquired data were analyzed using ProcartaPlex Analysis software v1.0.
6
Quantification of Cytokines and Proteins in Biological Samples
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IL-22 in human samples was quantified using either Human IL-22 Quantikine ELISA Kit (D2200, R&D Systems) or SMCTM Human IL-22 High Sensitivity Immunoassay Kit (EMD Millipore, 03-0162-00) according to manufacturers’ instructions. The SMC assay was read on a SMC Pro (EMD Millipore) instrument. The lower limit of quantification (LLOQ) of this immunoassay is 0.1 pg/mL. IL-22 in mouse samples was quantified using ELISA MAXTM Deluxe Set Mouse IL-22 (BioLegend, 436304). The LLOQ of this immunoassay is 3.9 pg/mL. S100A8 in human samples was quantified using Human S100A8 DuoSet ELISA (DY4570, R&D Systems).
Concentration of lineage-associated cytokines in cell culture supernatants of polarized T cells were quantified using a custom-made ProCarta Plex assay (Thermo Fisher Scientific) acquired on a Luminex platform. Hepcidin in mouse serum was quantified using a colorimetric assay from Hepcidin Murine-Compete ™ ELISA Kit from Intrinsic LifeSciences (HMC-001).
Concentration of lineage-associated cytokines in cell culture supernatants of polarized T cells were quantified using a custom-made ProCarta Plex assay (Thermo Fisher Scientific) acquired on a Luminex platform. Hepcidin in mouse serum was quantified using a colorimetric assay from Hepcidin Murine-Compete ™ ELISA Kit from Intrinsic LifeSciences (HMC-001).
7
Quantification of Cytokines and Proteins in Biological Samples
8
Multiplex Cytokine Profiling of Cell Cultures
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A custom Procartaplex assay (Thermofisher, Horsham) was used to measure human CCL2, CCL7, CX3CL1, CSF2, CXCL1, CXCL5, CXCL8, IFNγ, IL1α, IL1β, IL6, OSM and TNF in cell culture supernatants according to manufacturer’s instructions. Briefly, antibody coated capture beads were vortexed, transferred to 96-well plates and washed before sequential incubation with culture supernatant samples using a dilution range of x2-20 and standards for 2 hours at room temperature, mixture of biotinylated detection antibodies for each analyte for 30 minutes at room temperature, and Streptavidin-PE, washing the beads after each incubation. Data was acquired on the a Bioplex 200 platform (Bio-Rad, Watford).
9
Cytokine Profiling of Tumor Microenvironment
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Tumors or 3-MCA injection sites were homogenized in 1 mL PBS−/− containing protease inhibitors (Complete-EDTA-free; Roche) and PMSF (1mM). Tissue homogenates were centrifuged at 14000 rpm for 30 min at 4°C and supernatants were stored at −20°C for cytokine analysis. Murine IFNγ, CCL2, CXCL1, CXCL2, CXCL12, M-CSF, G-CSF, TNFα, IL-22, IL-1β, IL-6, IL-12p70, VEGF, TGFβ and IL-23p19 were measured in tissue homogenates by ELISA (R&D DuoSet ELISA Development System) according to manufacturer’s instructions. Murine IL-18, IL-17A were measured in tissue homogenates by ProcartaPlex Assay (ThermoFisher) according to manufacturer’s instructions.
10
Multiplex Biomarker Profiling and Analysis
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A custom ProcartaPlex assay (Thermo Fisher Scientific) was used to measure levels of 15 Random forest models were performed with the ClassifyR, dplyr, devtools, S4Vectors and ranger packages in R. Feature selection was confirmed using random forest biomarker analysis in MetaboAnalyst V.5.0, where sensitivity and specificity values were derived from confusion matrices. MetaboAnalyst V.5.0 was used for partial-least squares discriminant analyses (PLS-DA) and
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