Mice blood was centrifuged at 4000 g for 20 min at 4°C and sera were collected and frozen at −80°C. The concentrations of human and murine cytokines were quantified by electrochemiluminescence using multiplex assay kits from Meso Scale Discovery. Briefly, 25 μl of serum were added to the 96-well multi-array plates and the assays were processed following the manufacturer’s instructions. Plates were read on the multiplexing imager Sector S600 (Meso Scale Discovery).
Multiplex assay kit
Manufactured by Mesoscale
Sourced in United States
Multiplex assay kits are laboratory equipment designed to simultaneously measure multiple target analytes in a single sample. These kits provide a platform for the simultaneous detection and quantification of various biomolecules, such as proteins, nucleic acids, or metabolites, in a high-throughput and efficient manner.
Automatically generated - may contain errors
6 protocols using multiplex assay kit
1
Multiplex Cytokine Quantification in Mice Sera
2
Immune Cell Profiling in Tumor and Spleen
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Serum chemokine and cytokine levels were measured using customized Multiplex
Assay Kits (Meso Scale Discovery, Rockville, MD). Tumor and spleen immune cell
profiling was performed using a published method.16 Briefly, spleen and tumor tissues were collected and placed in plain 1×
HBSS. For isolation of lymphocytes from spleens and tumors, protocols described
by Dorta et al were used.16 In each sample, 1 × 106 cells were used for staining for
immune cell surface markers. Cells were then incubated at 4°C for 1 hour with
antibodies against mouse CD4 (BioLegend, San Diego, CA), CD3 (BD Biosciences,
San Jose, CA), CD8 (BioLegend), CD19 (BD Biosciences), and CD25 (BioLegend).
Subsequently, the cells were washed twice with PBS containing 2% FBS and then
fixed and permeabilized with Foxp3 Fix/Perm Kit (ThermoFisher Scientific,
Waltham, MA). Then cells were washed twice with wash buffer and incubated with
intracellular markers: Foxp3 (eBioscience, San Diego, CA), IFN-γ (BioLegend) for
1 hour at 4°C. Antibodies were diluted according to the manufacturers’
recommendations. All the samples were collected on a BD LSRFORTESSA X-20 (BD
Biosciences) and analyzed using FlowJo software (FlowJo v.10, Ashland, OR).
Assay Kits (Meso Scale Discovery, Rockville, MD). Tumor and spleen immune cell
profiling was performed using a published method.16 Briefly, spleen and tumor tissues were collected and placed in plain 1×
HBSS. For isolation of lymphocytes from spleens and tumors, protocols described
by Dorta et al were used.16 In each sample, 1 × 106 cells were used for staining for
immune cell surface markers. Cells were then incubated at 4°C for 1 hour with
antibodies against mouse CD4 (BioLegend, San Diego, CA), CD3 (BD Biosciences,
San Jose, CA), CD8 (BioLegend), CD19 (BD Biosciences), and CD25 (BioLegend).
Subsequently, the cells were washed twice with PBS containing 2% FBS and then
fixed and permeabilized with Foxp3 Fix/Perm Kit (ThermoFisher Scientific,
Waltham, MA). Then cells were washed twice with wash buffer and incubated with
intracellular markers: Foxp3 (eBioscience, San Diego, CA), IFN-γ (BioLegend) for
1 hour at 4°C. Antibodies were diluted according to the manufacturers’
recommendations. All the samples were collected on a BD LSRFORTESSA X-20 (BD
Biosciences) and analyzed using FlowJo software (FlowJo v.10, Ashland, OR).
3
Plasma Biomarker Measurement Protocol
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Plasma levels of total GLP-1, PYY, total ghrelin, insulin, and leptin were measured by multiplex assay kits based on chemiluminescence detection and following the manufacturer’s instructions (Meso Scale Discovery, Gaithersburg, MD). Analyses were done using a QuickPlex SQ 120 instrument (MSD) and DISCOVERY WORKBENCH®4.0 software.
4
Cytokine and Chemokine Profiling of MIS-C, pCOVID-19, and Healthy Controls
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Plasma samples from 14 patients with MIS-C, 14 patients with pCOVID-19, and 35 pHCs were measured, in duplicate, for 36 different cytokines (IFN-γ, IFN-α2a, IFN-β, IFN-λ1, IP-10, IL-10, IL-12p70, IL-13, IL-15, IL-16, IL-17A, IL-1α, IL-RA, IL-1β, IL-2, IL-2Rα, IL-23p40, IL-4, IL5, IL-6, IL-7, TNF-α, TGF-β GM-CSF and VEGF) and chemokines (IL-8, MIP-1α, MIP-1β, MCP-1, MCP-2, MCP-4, eotaxin, eotaxin 3, MDC, TARC) using the multiplex assay kit from Meso Scale Discovery (MSD) according to the manufacturer’s instructions. Data were acquired with a MESO QuickPlex SQ 120 instrument, and if the cytokine values were below the background, the values were set to 0.
5
Inflammatory Cytokine Assessment via ELISA
6
Cardiac Puncture Blood Collection and Analysis
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A cardiac puncture was used to collect blood in EDTA-coated tubes (Sarstedt Inc., Newton, NC; Microvette CB 300) on wet ice until centrifugation at 1 500 × g for 10 min at 4°C. The plasma supernatant was collected and stored at −80°C until analysis with a multiplex assay kit (Meso Scale Discovery, Rockville, MD) according to the manufacturer’s recommended protocols.
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