Human Perforin ELISpotPLUS kit (ALP) (CAT#: ITS-0322-P62)

2 plates

1.2 Pre-coated plates, mAb Pf-80/164.
2.Vial(blue top):Biotinylated detection mAb Pf-344(40μL);Concentration 0.5mg/ml.
3.Vial 2(white top):Streptavidin-ALP(40μL).
4.BCIP/NBT-plus substrate(25ml):The detection antibody is supplied in sterile filtered(0.2μm)PBS with 0.02% sodium azide. Streptavidin-ALP is supplied in 0.1M Tris buffer with 0.002% Kathon CG. Vials have been overfilled 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 cellsand stimuli can be mixed before addition to the plate.
4.Put the plate in a 37°Chumidified 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(MT114-biotin) to 0.05 ug/ml in PBS containing 0.5% fetal calfserum(PBS-O.5%FCS).Add 100 ul/well and incubate for 2 hours at room temperature.
7.Wash plate as above (step C1).
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 (step C1).
10.Filter the ready-to-use substrate solution(BCIP/NBT-plus) through a 0.45 um filter and add100 ul/well. Develop until distinct spots emerge.
11.Stop color development by washing extensively in tap water. If desirable, remove the underd-rain (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 micro-scope.
13.Store plate in the dark at room temperature.

human, non-human primates

Biotinylated detection mAb (Pf-344),Streptavidin-ALP,Substrate (BCIP/NBT-plus),Pre-coated MSIP white plates (mAbs Pf-80/164)

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 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.

FLH2; P1; PFN1; Cytolysin; HPLH2; PFP; Lymphocyte pore-forming protein; Perforin-1

Perforin

Perforin with structural similarities to complement component C9 that is important in immunity. This protein forms membrane pores that allow the release of granzymes and subsequent cytolysis of target cells. Whether pore formation occurs in the plasma membrane of target cells or in an endosomal membrane inside target cells is subject to debate. Mutations in this gene are associated with a variety of human disease including diabetes, multiple sclerosis, lymphomas, autoimmune lymphoproliferative syndrome (ALPS), aplastic anemia, and familial hemophagocytic lymphohistiocytosis type 2 (FHL2), a rare and lethal autosomal recessive disorder of early childhood.

5551

P14222

Among its related pathways are Allograft rejection and Granzyme Pathway.

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.