Mouse CCL5/RANTES ELISpot Development Module, 5 Plate 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.
* Sold separately (see Other Reagents Required, below)
** Following an overnight incubation with capture antibody, 2 hour blocking step, and cell stimulation.
*** When using the protocol provided.
Complete ELISpot kits are highly sensitive, microplate-based assays for the detection of cytokine secreting cells. Kits are available for detection and enumeration of a single analyte or two analytes simultaneously. Complete ELISpot kits are ready-to-run and require no assay development or refinement. ELISpot Development Modules contain the basic components required to develop an ELISpot assay. They offer an economical alternative to buying separate antibodies.
ELISpot development modules are an alternative to ELISpot kits. A basic understanding of ELISpot assay development is required for the successful use of these reagents. Each investigator should optimize the coating conditions, the assay sensitivity, the type of enzyme and substrate, as well as the concentrations of the capture and detection antibodies to achieve desired results. The analyte-specific ELISpot Development Module and the ELISpot Blue Color Module contain the necessary components for analyte detection and visualization, respectively. These modules can be used together but are sold separately. Each module contains enough reagents for at least five 96-well microplates.
- An economical alternative to ELISpot Kits
- Optimized capture and detection antibody pairings and recommended concentrations save lengthy development time
- Generic development protocols provide direction to start an optimization protocol
- Customize the assay to your specific needs
- Mouse CCL5/RANTES Capture Antibody
- Mouse CCL5/RANTES Biotinylated-conjugated Detection Antibody
OTHER REAGENTS REQUIRED
- ELISpot Blue Color Module or equivalent (R&D Systems, Catalog # SEL002)
- PBS - 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.2 - 7.4, 0.2 µm filtered
- Wash Buffer - 0.05% Tween® 20 in PBS
- Blocking Buffer - 1% BSA, 5% Sucrose in PBS
- Reagent Diluent - 1% BSA in PBS, pH 7.2 - 7.4, 0.2 µm filtered
- 2 °C – 8 °C refrigerator
- 37 °C CO2 incubator
- Positive Control - Use Recombinant Mouse CCL5/RANTES or cells known to secrete Mouse CCL5/RANTES
- 96-well plates - Nitrocellulose-bottom plates, PVDF-bottom Immunospot® plates, or flat-bottom polystyrene Immulon® ELISA plates
- Multi-channel pipette, squirt bottle, manifold dispenser, or automated microplate washer
- Dissection microscope or an automated ELISpot Reader
- Deionized H2O
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Preparation and Storage
CCL5/RANTES is a secreted beta-chemokine that plays a primary role in the inflammatory immune response through chemoattraction and activation of leukocytes that express CCR1, 3, 4, or 5. The two N-terminal residues of CCL5 can be removed by CD26/DPPIV, generating a protein that functions as a chemotaxis inhibitor. The deposition of CCL5 on activated vascular endothelial cells is crucial for monocyte adhesion to damaged vasculature. CCL5 is upregulated in breast cancer and promotes tumor progression through the attraction of proinflammatory macrophages in addition to its actions on tumor cells, stromal cells, and the vasculature. In humans, CCR5 binding to CCL5 inhibits the infectivity of R5 (M-tropic) but not X4 (T-tropic) strains of HIV-1.
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