A Multiplex Bead-Based Immunoassay to Quantify Multiple Cytokine Targets

Published: April 30, 2024

Abstract

Source: Lehmann, J. S., et al. Multiplex Cytokine Profiling of Stimulated Mouse Splenocytes Using a Cytometric Bead-based Immunoassay Platform. J. Vis. Exp. (2017).

This video demonstrates a multiplex bead-based immunoassay for quantifying multiple cytokine targets in a sample. A mixture of distinct microspheres conjugated to cytokine-specific antibodies is used to bind the cytokines present in the sample. Streptavidin-tagged reporters are immobilized on the bound-cytokines using biotinylated detection antibodies. Flow cytometry identifies the bound cytokines based on the unique sizes and internal fluorescence intensities of the microspheres, while the fluorescence intensity of the reporters indicates the number of cytokines.

Protocol

1. Biological Sample Preparation NOTE: The chosen anatomical site and volume of blood collection are left to the discretion of each individual investigator and will not affect the outcome of the assay. However, once they are obtained, samples should be handled according to the steps listed below. Preparation of Serum Samples Collect the desired volume of blood into the preferred collection tube and allow it to clot for …

Divulgaciones

The authors have nothing to disclose.

Materials

Allegra 6R Centrifuge with MICROPLUS Carrier Adaptor Beckman Coulter 366816 For LEGENDplex assays is run in microtubes, V-or U-bottom 96-well plate (optional)
LEGENDplex Mouse T helper Cytokine Panel BioLegend 740005 Multiplex Immunoassay (13-plex)
Anti-mouse CD3ε antibody BioLegend 100314 Clone 145-2C11, low endotoxin, azide free format
Anti-mouse CD28 antibody BioLegend 102112 Clone 37.51, low endotoxin, azide free format
Vacuum Pump EMD Millipore WP6111560 For LEGENDplex assays using filter plates (recommended)
Vacuum Manifold EMD Millipore MSVMHTS00 For LEGENDplex assays using filter plates (recommended)
Sterile Disposable Reagent Reservoirs Fisher Scientific 07-200-130 Or equivalent
Polypropylene MicroFACS Tubes Fisher Scientific 11-842-90 For LEGENDplex assays is run in microtubes, V-or U-bottom 96-well plate (optional)
Pipette Kit Fisher Scientific 14-388-100 Four pipette sizes (0.2-2µL, 2-20µL, 20-200µL, 100-1000µL)
Multi-Channel Pipette Fisher Scientific FA10011G capable of dispensing 5 μL to 200 μL
Microplate Shaker Fisher Scientific 88880023 Or equivalent
Microcentrifuge Fisher Scientific 75002410 Or equivalent
FACS tubes Fisher Scientific NC9885747 12 x 75 mm round bottom
PMA (Phorbol 12-myristate 13-acetate) Sigma-Aldrich P8139
Lipopolysaccharide (LPS) Sigma-Aldrich L-8274 Escherichia coli O26:B6
Ionomycin Sigma-Aldrich I0634
Branson B200 Ultrasonic Cleaner Sigma-Aldrich Z305359 Or equivalent
Microcentrifuge tubes Sigma-Aldrich Z666505 1.5 mL polypropylene tubes
Flow Cytometer Various Various Cytometer equipped with single laser (488nm blue) or two lasers (488nm blue or 532nm green + 633nm Red) capable of reading emission wavelengths at 575nm and 660nm
96-Well Polypropylene Plate VWR 82050-662 For LEGENDplex assays is run in microtubes, V-or U-bottom 96-well plate (optional)

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A Multiplex Bead-Based Immunoassay to Quantify Multiple Cytokine Targets. J. Vis. Exp. (Pending Publication), e22121, doi: (2024).

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