Human IL-12 (p70) ELISpotPLUS kit (ALP) (CAT#: ITS-0322-P116)

2 plates

1.2 pre-coated plates, mAb MT86/221.
2.Vial 1 (blue top):Biotinylated detection mAb MT704 (40 μl);Concentration 0.5 mg/ml.
3.Vial 2 (white top):Streptavidin-HRP (40 μl).
4.TMB substrate (25 ml):The detection antibody is supplied in sterile fltered (0.2um) PBS with 0.02% sodium azide. Streptavidin-HRP is supplied in PBS with 0.002% Kathon CG. Vials have been overflled to ensure recovery of the specified amount.

200 µL

2h

Pre-coated

1.Remove the plate from the sealed package and wash 4 times with sterile PBS(200 ul/well).
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. Do not move the plate during this time and take measures to avoid evaporation.
5.Remove the cells by emptying the plate and wash 5 times with PBS, 200 ul/well.
6.Dilute the detection antibody(MT704-biotin) to 0.05 ug/ml in PBS containing 0.5% fetal calf serum(PBS-O.5%FCS). Add 100 ul/well and incubate for 2 hours at room temperature.
7.Wash plate as above.
8.Dilute the Streptavidin-ALP(1:1000) in PBS-0.5% FCS and add 100 ul/well. Incubate for 1 hour at room temperature.
9.Wash plate as above.
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. If desirable, remove the underdrain (the soft plastic under the plate) and rinse the underside of the membrane.
12.Leave the plate to dry. Inspect and count spots in an ELISpot reader or in a dissection microscope.13. Store plate in the dark at room temperature.

human, non-human primates

Biotinylated detection mAb (MT704), Streptavidin-ALP, Substrate (BCIP/NBT-plus), Pre-coated MSIP white plates (mAbs MT86/221)

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 (0.3 um) for optimal results. Although possible to use, we do not 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.

CLMF; Cytotoxic lymphocyte maturation factor 35 kDa subunit; NFSK; NK cell stimulatory factor chain 1; CLMF p35; NKSF1; IL-12 subunit p35; IL-12A; Interleukin-12 subunit alpha; P35

CLMF, IL-12A, NFSK, NKSF, NKSF2, NKSF1, CLMF2, P35, IMD29, IMD28, IL-12B

This gene encodes a subunit of a cytokine that acts on T and natural killer cells, and has a broad array of biological activities. The cytokine is a disulfide-linked heterodimer composed of the 35-kD subunit encoded by this gene, and a 40-kD subunit that is a member of the cytokine receptor family. This cytokine is required for the T-cell-independent induction of interferon (IFN)-gamma, and is important for the differentiation of both Th1 and Th2 cells. The responses of lymphocytes to this cytokine are mediated by the activator of transcription protein STAT4. Nitric oxide synthase 2A (NOS2A/NOS2) is found to be required for the signaling process of this cytokine in innate immunity.

3593

P29460

Among its related pathways are Tuberculosis and Proteoglycans in cancer.

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.