* 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.
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
Biotinylated Detection Antibody
Streptavidin conjugated to Alkaline Phosphatase
Wash Buffer Concentrate
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
IFN-gamma (Interferon-gamma) is the prototype proinflammatory cytokine and is produced by a variety of immune cells under inflammatory conditions, notably by T cells and NK cells. It plays a key role in host defense by promoting the development and activation of Th1 cells, chemoattraction and activation of monocytes and macrophages, upregulation of antigen presentation molecules, and immunoglobulin class switching in B cells. It also exhibits antiviral, antiproliferative, and apoptotic effects. In addition, IFN-gamma functions as an anti-inflammatory mediator by promoting the development of regulatory T cells and inhibiting Th17 cell differentiation. IFN-gamma dimers signal through a receptor complex of two IFN-gamma R1 and two IFN-gamma R2 subunits.
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.
Phase I therapeutic trial of the HIV-1 Tat protein and long term follow-up. Authors: Longo O, Tripiciano A, Fiorelli V, Bellino S, Scoglio A, Collacchi B, Alvarez MJ, Francavilla V, Arancio A, Paniccia G, Lazzarin A, Tambussi G, Din CT, Visintini R, Narciso P, Antinori A, D'Offizi G, Giulianelli M, Carta M, Di Carlo A, Palamara G, Giuliani M, Laguardia ME, Monini P, Magnani M, Ensoli F, Ensoli B Vaccine, 2009;27(25):3306-12. Species: Human Sample Type: Whole Cells