Murine IL-2 ELISpot Kit (CAT#: ITS-0322-P11)

1x96 Wells

1.Pre-coated 96 well PDVF bottomed plates (5).
2.Biotinylated detection antibody (lyophilised, resuspend in 0.55ml).
3.Streptavidin-Alkaline Phosphatase conjugate (50µl).
4.Bovine Serum Albumin (BSA).
5.Ready to use BCIP/NBT substrate buffer (50ml).

1.Miscellaneous laboratory plastic and/or glass, if possible sterile.
2.Cell culture reagents.
3.Cell stimulation reagents (PMA, Ionomycin).
4.CO2 incubator.
5.Tween 20.
6.Phosphate Buffered Saline (PBS).

100 µL

1.5h

Pre-coated

1.1X Phosphate Buffered Saline (PBS).
2.1% BSA PBS Solution (Dilution Buffer):For one plate dissolve 0.2 g of BSA in 20 ml of 1X PBS.
3.0.05% PBS-T Solution (Wash Buffer):For one plate dissolve 50µl of Tween 20 in 100mL of 1X PBS.
4.Detection Antibody:Reconstitute the lyophilised antibody with 0.55mL of distilled water. Gently mix the solution and wait until all the lyophilised material is back into solution.
5.Streptavidin - AP conjugate:For 1 plate dilute 10µl of Streptavidin-AP conjugate into 10 mL Dilution Buffer and mix well.

1.Add 100µl of 1X PBS to every well.
2.Incubate plate at room temperature (RT) for 10 min.
3.Empty the wells by flicking the plate over a sink & gently tapping on absorbent paper.
4.Add 100µl of sample, positive and negative controls cell suspension to appropriate wells providing the required concentration of cells and stimulant.
5.Cover the plate and incubate at 37oC in a CO2 incubator for an appropriate length of time (15-20 hours).
6.Empty the wells and remove excess solution then add 100µl of PBS-T to every well.
7.Incubate the plate at 4oC for 10 min.
8.Empty the wells as previous and wash the plate 3x with 100µl of PBS-T.
9.Add 100µl of diluted detection antibody to every well.
10.Cover the plate and incubate at RT for 1 hour 30 min.
11.Empty the wells as previous and wash the plate 3x with 100µl of PBS-T.
12.Add 100µl of diluted Streptavidin-AP conjugate to every well.
13.Cover the plate and incubate at RT for 1 hour.
14.Empty the wells and wash the plate 3x with 100µl of PBS-T.
15.Peel of the plate bottom and wash both sides of the membrane 3x under running distilled water, once washing complete remove any excess solution by repeated tapping on absorbent paper.
16.Add 100µl of ready-to-use BCIP/NBT buffer to every well.
17.Incubate the plate for 5-15 min monitoring spot formation visually throughout the incubation period to assess sufficient colour development.
18.Empty the wells and rinse both sides of the membrane 3x under running distilled water. Completely remove any excess solution by gentle repeated tapping on absorbent paper.

Murine

CAA07317.1

Liquid

1.For research use only not to be used as a diagnostic test.
2.Do not eat, drink, smoke or apply cosmetics where kit reagents are used.
3.Cover or cap all reagents when not in use.
4.Do not mix or interchange reagents between different lots.
5.Do not use reagents beyond the expiration date of the kit.
6.When pipetting reagents, maintain a consistent order of addition from well-to-well. This will ensure equal incubation times for all wells.
7.BCIP/NBT buffer is potentially carcinogenic and should be disposed of appropriately, caution should be taken when handling this reagent, always wear gloves.

1.Handling of reagents, serum or plasma specimens should be in accordance with local safety procedures.
2.When not in use, kit components should be stored refrigerated or frozen as indicated on vials or bottles labels.
3.All reagents should be warmed to room temperature before use.
4.Use a clean disposable plastic pipette tip for each reagent, standard, or specimen addition in order to avoid cross contamination.
5.Use a clean plastic container to prepare the washing solution.
6.Thoroughly mix the reagents and samples before use by agitation or swirling.

Store kit reagents between 2 and 8°C.

Immediately after use remaining reagents should be returned to cold storage (2 to 8°C). The expiry of the kit components can only be guaranteed if the components are stored properly, and if in the case of repeated use of one component, the reagent is not contaminated by the first handling.

Expiry of the kit and reagents is stated on box front labels.

Select online data sheet information is drawn from bioinformatics databases, occasionally resulting in ambiguous or non-relevant product information. It is the responsibility of the customer to review, verify, and evaluate the information to make sure it matches their requirements before purchasing the kit. Our ELISA Kit assays are dynamic research tools and sometimes they may be updated and improved.

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

TCGF; IL-2; lymphokine; Interleukin-2; Aldesleukin; T-cell growth factor

IL-2; TCGF; lymphokine

The protein encoded by this gene is a secreted cytokine that is important for the proliferation of T and B lymphocytes. The receptor of this cytokine is a heterotrimeric protein complex whose gamma chain is also shared by interleukin 4 (IL4) and interleukin 7 (IL7). The expression of this gene in mature thymocytes is monoallelic, which represents an unusual regulatory mode for controlling the precise expression of a single gene. The targeted disruption of a similar gene in mice leads to ulcerative colitis-like disease, which suggests an essential role of this gene in the immune response to antigenic stimuli.

3558

P60568

Among its related pathways are Allograft rejection and PEDF Induced Signaling.

1.96-PVDF bottomed-well plates are first treated with 35% ethanol and then coated with capture antibody.
2.Incubation of cells in the coated microwell.
3.Cell removal by washing. Incubation with biotinylated antibody.
4.Incubation with streptavidin - alkaline phosphatase conjugated.
5.Addition of substrate BCIP/NBT and monitoring of spot formation.