Bovine IL-4 ELISpotPLUS kit (ALP)

This kit is ideal for users who want convenient and sensitive analysis.

The purpose of this experiment is to count bovine IL-4 secreting cells. The kit includes monoclonal antibodies, biotinylated detection antibodies, Streptavidin-ALP, and ELISpot plates pre-coated with BCIP/NBT-plus substrates.

ELISpot

Washing of plates can be done using a multi-channel micropipette. In washing steps not requiring sterile conditions(C1-C5), a regular ELISA plate washer can also be used, provided that the washing head is adapted to the ELISpot plates.

IL4

cow, sheep

Sandwich

Sandwich

cell suspension

Recognizes natural bovine IL-4

The monoclonal antibodies in this kit cross-react with IL-4 from sheep.

2 plates

1.2 Pre-coated plates, mAb bIL4-I
2.Vial(blue top): Biotinylated detection mAb bIL4-II(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, 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(26E5-biotin) to 0.5 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.

Bovine

Biotinylated detection mAb (bIL4-II), Streptavidin-ALP, Substrate (BCIP/NBT-plus), Pre-coated MSIP white plates (mAb bIL4-I)

The number of cells responding to stimulation is often compared to the number of cells spontaneously producing the cytokine, which is determined by incubating the same number of cells in the absence of stimuli. A polyclonal activator such as phytohemagglutinin or concanavalin A (1-10 ug/ml) is often included as a control for cell viability and functionality of the test system.

PBS for washing and dilution should be filtered (0.2 um) for optimal results. Avoid the inclusion of Tween or other detergents in the washing and incubation buffers.
To reduce unspecific background it is recommended to filter (0.2 um) the working dilution of detction mAb.

4°C/-20°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.

BSF-1; IL-4; B-cell stimulatory factor 1; BCGF1; Lymphocyte stimulatory factor 1; BCGF-1; Interleukin-4; Pitrakinra; Binetrakin; BSF1

IL-4

The protein encoded by this gene is a pleiotropic cytokine produced by activated T cells. This cytokine is a ligand for interleukin 4 receptor. The interleukin 4 receptor also binds to IL13, which may contribute to many overlapping functions of this cytokine and IL13. STAT6, a signal transducer and activator of transcription, has been shown to play a central role in mediating the immune regulatory signal of this cytokine. This gene, IL3, IL5, IL13, and CSF2 form a cytokine gene cluster on chromosome 5q, with this gene particularly close to IL13. This gene, IL13 and IL5 are found to be regulated coordinately by several long-range regulatory elements in an over 120 kilobase range on the chromosome. Two alternatively spliced transcript variants of this gene encoding distinct isoforms have been reported.

280824

P30367

Among its related pathways are Notch Signaling Pathway and Allograft rejection.

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.