Mouse IFN-gamma ELISpot PLUS kit (ALP), strips (CAT#: ITS-0322-P41)

10 plates

1.10 pre-coated strip plates, mAb AN18.
2.Empty plate frame included for transfer of strips.
3.Vial 1 (yellow top):Biotinylated detection mAb R4 6A2 (150 μul);Concentration 1 mg/ml.
4.Vial 2 (white top):Streptavidin-ALP (150 ul).
5.BCIP /NBT-plus substrate (120 ml):The detection antibody is supplied in sterile filtered (0.2um) PBS with 0.02% sodium azide. Streptavidin-ALP is supplied in 0.1 M Tris buffer with 0.002% Kathon CG. Vials have been overfilled to ensure recovery of the specifed amount.

200 µL

2h

Pre-coated

1.Assemble the required number of strips in the extra plate frame and wash 4 times with sterile PBS (200 yl/well). Seal the bag with the remaining strips and store at room temperature.
2.Condition the plate with medium (200 ul/well) containing 10% of the same serum as used for the cell suspensions. Incubate for at least 30 minutes at room temperature.
3.Remove the medium and add the stimuli followed by the cell suspension. Alternatively cells and stimuli can be mixed before addition to the plate.
4.Put the plate in a 37°C humidified incubator with 5% CO, and incubate for 12-48 hours.
5.Remove the cells by emptying the plate and wash 5 times with PBS, 200 ul/well.
6.Dilute the detection antibody (R4-6A2-biotin) to 1 ug/ml in PBS containing 0.5% fetal calf serum (PBS-0.5% FCS). Add 100 ul/well and incubate for 2 hours at room temperature.
7.Wash plate as above (stepC1).
8.Dilute the Streptavidin-ALP (1:1000) in PBS-0.5% FCS and add 100 yl/well. Incubate for 1 hour at room temperature.
9.Wash plate as above (step C1).
10. Filter the ready-to-use substrate solution (BCIP/NBT-plus) through a 0.45 um filter and add 100 ul/well. Develop until distinct spots emerge.
11.Stop color development by washing extensively in tap water. Remove the plate frame from the plastic tray and rinse the underside of the membranc.
12.Leave the plate to dry. Inspect and count spots in an ELISpot reader or in a disection microscope.
13.Store plate in the dark at room temperature.

Mouse

Biotinylated detection mAb (R4-6A2),Streptavidin-ALP,Substrate (BCIP/NBT-plus),Pre-coated strip clear plates (mAb AN18)

We recommend the use of PVDF-based membrane plates. Maximal antibody binding capacity of these platesis obtained by a brief treatment with ethanol.

PBS for washing and dilution should be filtered (0.2 um) for optimal results.Although possible to use, we donot recommend the inclusion of Tween or other detergents in the washing and incubation buffers.

4°C-8°C

Plates should be kept at room temperature.

The expiry date indicates how long unopened products, stored according to instructions, are recommended for use.

The serum should be selected to support cell culture and give low background staining. We recom-mend the use of fetal calf serum. Alternatively serum-free medium evaluated for cell culture can beused.

For Research Use Only. Not for use in diagnostic procedures.

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

IFNG2

This gene encodes a soluble cytokine that is a member of the type II interferon class. The encoded protein is secreted by cells of the both the innate and adaptive immune systems. The active protein is a homodimer that binds to the interferon gamma receptor which triggers a cellular response to viral and microbial inflections. Mutations in this gene are associated with an increased susceptibility to viral, bacterial and parasitic infections and to several autoimmune diseases.

3458

P01579

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

1.Antibody coating:Cytokine-specific monoclonal capture antibodies are immobilized on an ethanol-treated PVDF membrane plate.
2.Cell incubation:Cells are added to the wells in the presence or absence of activating stimuli, and then incubated to allow for cytokine secretion.
3.Cytokine capture:Secreted cytokines bind to the capture antibodies on the membrane immediately surrounding the activated cells.
4.Detection antibodies:Following removal of the cells and washing of the plate wells, biotinylated cytokine-specific detection antibodies are added to the wells.
5.Streptavidin-enzyme conjugate:To enable the formation of spots on the membrane, a streptavidin-enzyme conjugate is added to the wells.
6.Addition of substrate:Colorimetric substrate is added to the wells and will form an insoluble precipitate when catalyzed by the enzyme; a visible representation of cytokine release by a single activated cell.
7.Analysis Spots are counted in an automated ELISpot reader or under a dissection microscope, and the frequency of secreting cells is calculated.