* Following cell stimulation.

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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.

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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

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KIT CONTENTS

• Microplate
• Biotinylated Detection Antibody
• Streptavidin conjugated to Alkaline Phosphatase
• Dilution Buffers
• Wash Buffer Concentrate
• BCIP/NBT Chromogen
• Positive Control

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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: IFN-gamma

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.