Feline IFN-gamma ELISpot Development Module, 5 Plate

The complete ELISpot kit is highly sensitive, a microplate-based method for detecting cytokine secreting cells. The kit can be used to detect and enumerate one or two analytes simultaneously.

The complete ELISpot kit is ready to run and does not require analytical development or improvement. The enzyme-linked immunosorbent assay module contains the basic components required for enzyme-linked immunosorbent assay. They provide an economical option for purchasing individual antibodies.

1.An economical alternative to ELISpot Kits.
2.Optimized capture and detection antibody pairings and recommended concentrations save lengthy development time.
3.Generic development protocols provide direction to start an optimization protocol.
4.Customize the assay to your specific needs.

ELISpot

When a 96-well PVDF microplate is used, 1:60 dilutions of the provided capture and detection antibodies are recommended. Each investigator should determine the optimal working dilution of the antibodies depending on the type of microplate, Wash Buffer, and Blocking Buffer used.

IFN-gamma

Feline

Sandwich

Quantitative Sandwich ELISA Development Module

Whole Cells

anti-feline IFN-γ monoclonal antibody. ∗

1 Kit (for 5 Plates)

1.Feline IFN-gamma Capture Antibody.
2.Feline IFN-gamma Biotinylated-conjugated Detection Antibody.

1.ELISpot Blue Color Module or equivalent.
2.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.
3.Wash Buffer - 0.05% Tween 20 in PBS.
4.Blocking Buffer - 1% BSA, 5% Sucrose in PBS.
5.Reagent Diluent - 1% BSA in PBS, pH 7.2 - 7.4, 0.2 µm filtered.
6.2 °C - 8 °C refrigerator.
7.37 °C CO2 incubator.
8.Positive Control - Use Recombinant Human IgG B Cell ELISpot Development Modules or cells known to secrete Human IgG B Cell ELISpot Development Modules.
9.96-well plates - Nitrocellulose-bottom plates, PVDF-bottom ELISAspot plates, or flat-bottom polystyrene ELISA plates.
10.Multi-channel pipette, squirt bottle, manifold dispenser, or automated microplate washer.
11.Dissection microscope or an automated ELISpot Reader.
12.Deionized H2O.

100 µL

3 hours 35 mins to 4 hours 50 mins**

Pre-coated

1.Capture Antibody Concentrate: Reconstitute with 1 mL of PBS. After reconstitution, store at 2-8 °C for up to 60 days or aliquot and store at -20 °C to -70 °C in a manual defrost freezer for up to 6 months.
2.Detection Antibody Concentrate: Reconstitute with 1 mL of Reagent Diluent. After reconstitution, store at 2-8 °C for up to 60 days or aliquot and store at -20 °C to -70 °C in a manual defrost freezer for up to 6 months.

Feline

96-well microplate, sold separately

Avoid inhalation, contact with eyes, skin and clothing. Avoid the formation of dust and aerosols. Use in a well-ventilated area. Keep away from sources of ignition. Avoid prolonged or repeated exposure.

Use a manual defrost freezer and avoid repeated freeze-thaw cycles. Do not use past expiration date.

Store the unopened product at -20 to -70 °C.

Store in cool, well-ventilated area. Keep away from direct sunlight. Keep container tightly sealed until ready for use.

For Research Grade Use Only.

IFN-gamma; Interferon gamma; Immune interferon; IFG; IFI

IFNG2

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.

493965

P46402

Among its related pathways are Allograft rejection and IL1 and megakaryocytes in obesity.

1.Calculate the total volume of Capture Antibody needed and dilute to the working concentration in PBS.
2.Immediately add 100 µL of the diluted Capture Antibody or antigen per well. Cover the microplate with the lid and incubate overnight at 2-8 °C.
3.Aspirate each well and wash 3 times with Wash Buffer or PBS (350 µL/well) using a squirt bottle, manifold dispenser, or autowasher. After the final wash, remove any remaining liquid by inverting the microplate and blotting it against a clean paper towel.
4.Block membranes by adding 200 µL of Blocking Buffer to each well. Incubate for 2 hours at room temperature.
5.Aspirate or decant Blocking Buffer. Rinse with the same media in which the cells will be cultured.
6.Aspirate culture media from the microplate, and immediately fill the appropriate wells with 100 µL of culture media containing Feline IFN-gamma secreting cells. Incubate at 37 °C in a 5% CO2 incubator.
7.Wash the microplate 4 times with Wash Buffer. Remove any remaining Wash Buffer by inverting the microplate and blotting it against a clean paper towel.
8.Calculate the total volume of Detection Antibody needed and dilute to the working concentration in Reagent Diluent.
9.Add 100 µL of the diluted Detection Antibody or biotinylated antigen per well. Cover the microplate with the lid and incubate overnight at 2-8 °C.
10.Aspirate Detection Antibody or biotinylated antigen and wash as described in step 3. Microplates are now ready for color development.