Human Granzyme B ELISpot Kit Summary
* 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.
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 human Granzyme B. Complete ELISpot kits are ready-to-run and require no assay development or refinement.
This ELISpot assay employs a capture antibody specific for human Granzyme B, 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 human Granzyme B. 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 human Granzyme B secreting cell. The spots can be counted with an automated ELISpot reader system or manually using a stereomicroscope.
- Detect and quantitate individual cells secreting human Granzyme B
- 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
- Human Granzyme B Microplate
- Biotinylated Detection Antibody
- Streptavidin conjugated to Alkaline Phosphatase
- Dilution Buffers
- Wash Buffer Concentrate
- BCIP/NBT Chromogen
- Human Granzyme B 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: Granzyme B
Granzyme family serine proteases are stored in the granules of cytotoxic T lymphocytes and natural killer cells. Granzymes contain one S1 protease domain and are synthesized as preproproteins. Granzymes are released toward pathogen infected or transformed target cells during cellular immune reactions. Target cell entry by granzymes is through perforin channels. Granzymes trigger apoptosis by caspase-dependent and -independent mechanisms. There are five granzymes (A, B, G, H and K) in human and many more in mouse and rat.
Citation for Human Granzyme B ELISpot Kit
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
1 Citation: Showing 1 - 1
SHP-2 expression negatively regulates NK cell function.
Authors: Purdy AK, Campbell KS
J. Immunol., 2009;183(11):7234-43.
Sample Types: Whole Cells
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