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Assay Type
Quantitative Sandwich ELISA
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Assay Format
96-well PVDF-backed microplate
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Assay Length
3 hours 15 mins to 4 hours 30 mins*
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Sample Type
Whole Cells
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Specificity
Please see the
* Provided that the recommended microplates, buffers, diluents, substrates and solutions are used, and the assay is run as summarized in the Assay Procedure provided.
* Following cell stimulation.
PRODUCT SUMMARY
ELISpot kits are highly sensitive, microplate-based assays for the detection of cytokine secreting cells. This kit is designed for the detection and enumeration of . Complete ELISpot kits are ready-to-run and require no assay development or refinement.
This ELISpot assay employs a capture antibody specific for , pre-coated onto a PVDF-backed microplate. Appropriately stimulated cells are pipetted directly into the wells and the immobilized antibody in the immediate vicinity of the secreting cells binds secreted . Following wash steps and incubation with a biotinylated detection antibody, alkaline-phosphatase conjugated to streptavidin is added. Unbound enzyme is subsequently removed by washing and a substrate solution (BCIP/NBT) is added. A blue-black colored precipitate forms at the sites of cytokine localization and appears as spots, with each individual spot representing an individual secreting cell. The spots can be counted with an automated ELISpot reader system or manually using a stereomicroscope.
PRODUCT FEATURES
- Detect and quantitate individual cells secreting
- High sensitivity - ELISpot assays can measure responses with frequencies well below 1 in 100,000 cells
- No in vitro expansion of cells required
- High-throughput - ELISpot assays use only a small number of primary cells
KIT CONTENTS
- Microplate
- Biotinylated Detection Antibody
- Streptavidin conjugated to Alkaline Phosphatase
- Dilution Buffers
- Wash Buffer Concentrate
- BCIP/NBT Chromogen
- Positive Control
OTHER REAGENTS REQUIRED
- Pipettes and pipette tips
- Deionized or distilled water
- Squirt bottle, manifold dispenser, or automated microplate washer
- 500 mL graduated cylinder
- 37 °C CO2 incubator
- Sterile culture media
- Dissection microscope or an automated ELISpot reader
Preparation and Storage
Background: IL-5
IL-5 (Interleukin-5) is a secreted cytokine that is expressed as a covalent dimer. It is produced by activated Th2 cells, eosinophils, mast cells, and iNKT cells and promotes eosinophilic and basophilic differentiation and activation. IL-5 signals through a receptor complex containing IL-5 R alpha and the Common beta Chain/CD131 on eosinophils and basophils. Naturally occurring soluble forms of IL-5 R alpha retain the ability to bind IL-5 and limit its bioavailability.
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Entrez Gene IDs:
3567 (Human); 16191 (Mouse); 24497 (Rat); 397409 (Porcine); 280825 (Bovine); 403790 (Canine); 493803 (Feline);
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Long Name:
Interleukin 5
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Aliases:
BCDF mu; B-cell differentiation factor I; BCGFII; EDF; Eo-CSF; Eosinophil differentiation factor; IL5; IL-5; IL-5T-cell replacing factor; interleukin 5 (colony-stimulating factor, eosinophil); interleukin-5; TRF; TRFB cell differentiation factor I