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 …
Açıklamalar
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)