Porcine IL-10 ELISpot Kit

The ELISpot kit is a highly sensitive, microplate-based method for detecting cytokine secreting cells. This kit is used for the detection and counting of pig IL-10. The complete ELISpot kit is ready to run and does not require analytical development or improvement.

The ELISpot detection uses pig IL-10 specific capture antibody, pre-coated on a microtiter plate supported by pvdf. The appropriately stimulated cells are pipetted directly into the wells, and the immobilized antibody near the secreting cells binds to the secreted porcine IL-10. Following washing steps and incubating with biotinylated detection antibody, alkaline phosphatase bound to streptavidin is added. Then the unbound enzyme is removed by washing, and the substrate solution (BCIP/NBT) is added. A blue-black precipitate was formed at the location of the cytokine in the form of spots, each spot representing a single pig IL-10 secreting cell. These spots can be counted manually with an automatic ELISpot reading system or with a stereo microscope.

1.Detect and quantitate individual cells secreting porcine IL-10.
2.High sensitivity - ELISpot assays can measure responses with frequencies well below 1 in 100,000 cells.
3.No in vitro expansion of cells required.
4.High-throughput - ELISpot assays use only a small number of primary cells.

ELISpot

The enzyme-linked immunospot (ELISpot) assay was originally developed for the detection of individual B cells secreting antigen-specific antibodies. This method has since been adapted for the detection of individual cells secreting specific cytokines or other antigens. ELISpot assays employ the quantitative sandwich enzyme-linked immunosorbent assay (ELISA) technique.
A monoclonal antibody specific for porcine IL-10 has been pre-coated onto a polyvinylidene difluoride (PVDF)-backed microplate. Appropriately stimulated cells are pipetted into the wells and the microplate is placed into a humidified 37 °C CO2 incubator for a specified period of time. During this incubation period, the immobilized antibody in the immediate vicinity of the secreting cells binds secreted IL-10. After washing away any cells and unbound substances, a biotinylated monoclonal antibody specific for porcine IL-10 is added to the wells. Following a wash to remove any unbound biotinylated antibody, alkaline- phosphatase conjugated to streptavidin is added. Unbound enzyme is subsequently removed by washing and a substrate solution (BCIP/NBT) is added. A blue-black colored precipitate forms and appears as spots at the sites of cytokine localization, with each individual spot representing an individual IL-10 secreting cell. The spots can be counted with an ELISpot reader system or using a stereomicroscope.

IL-10

Porcine

Sandwich

Quantitative Sandwich ELISA

Whole Cells

1 Kit

1.Porcine IL-10 Microplate.
2.Biotinylated Detection Antibody.
3.Streptavidin conjugated to Alkaline Phosphatase.
4.Dilution Buffers.
5.Wash Buffer Concentrate.
6.BCIP/NBT Chromogen.
7.Porcine IL-10 Positive Control.

1.Pipettes and pipette tips.
2.Deionized or distilled water.
3.Squirt bottle, manifold dispenser, or automated microplate washer.
4.500 mL graduated cylinder.
5.37 °C CO2 incubator.
6.Sterile culture media.
7.Dissection microscope or an automated ELISpot reader.

100 µL

3 hours 15 mins to 4 hours 30 mins*

Pre-coated

1.Wash Buffer - If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. To prepare Wash Buffer, add 50 mL of Wash Buffer Concentrate to 450 mL of deionized water and mix well.
2.Porcine IL-10 Positive Control - Reconstitute the lyophilized Porcine IL-10 Positive Control with 250 μL of culture medium that is used to incubate cells.
3.Detection Antibody Mixture - Tap or vortex the vial to release reagent collected in the cap. Transfer 100 μL of Porcine IL-10 Detection Antibody Concentrate into the vial labeled Dilution Buffer 1 and mix well.
4.Streptavidin-AP Concentrate A - Tap or vortex the vial to release reagent collected in the cap. Transfer 100 μL of Streptavidin-AP Concentrate A into the vial labeled Dilution Buffer 2 and mix well.

1.Fill all wells in the microplate with 200μL of sterile culture media and incubate for approximately 20 minutes at room temperature.
2.When cells are ready to be plated, aspirate the culture media from the wells. Immediately add 100μL of the appropriate cells or controls to each well.
3.Incubate cells in a humidified 37°C CO2 incubator. Optimal incubation time for each stimulus should be determined by the investigator.
4.Aspirate each well and wash, repeating the process three times for a total of four washes.
5.Add 100 μL of the diluted Detection Antibody Mixture into each well, and incubate overnight at 2-8°C. Alternatively, incubation with detection antibodies can be done for 2 hours at room temperature on a rocking platform.
6.Repeat the wash procedure described in step 4.
7.Add 100 μL of the diluted Streptavidin-AP Concentrate A into each well, and incubate for 2 hours at room temperature.
8.Repeat the wash procedure described in step 4.
9.Add 100μL of the BCIP/NBT Substrate into each well, and incubate for 1 hour at room temperature. Protect from light.
10.Decant the BCIP/NBT Substrate from the microplate and rinse the microplate with deionized water. Invert the microplate and tap to remove excess water. Remove the flexible plastic underdrain from the bottom of the microplate, wipe the bottom of the plate thoroughly with paper towels and dry completely either at room temperature (60-90 minutes) or 37 °C (15-30 minutes).

Porcine

96-well PVDF-backed microplate

Some components of this kit contain sodium azide, which may react with lead and copper plumbing to form explosive metallic azides. Flush with large volumes of water during disposal.
BCIP/NBT is toxic if swallowed, in contact with skin, or if inhaled. It is a highly flammable liquid and vapor may cause serious irritation and damage to organs. Do not eat, drink, or smoke when using this product. Do not breathe fumes. Use only in a well-ventilated area. Keep away from heat, sparks, open flames, and hot surfaces. Keep the container tightly closed.

Some components in this kit contain a preservative which may cause an allergic skin reaction. Avoid breathing mist.
Wear protective gloves, clothing, eye, and face protection. Wash hands thoroughly after handling. Refer to the SDS on our website prior to use.

Store the unopened kit at 2-8 °C.

Do not use past kit expiration date.

This kit is validated for single use only. Results obtained using previously opened or reconstituted reagents may not be reliable.

For Research Grade Use Only.

IL10A; GVHDS; TGIF; IL-10; Cytokine synthesis inhibitory factor; CSIF; Interleukin-10

CSIF, Il-10

Interleukin-10 functions as an anti-inflammatory cytokine by inhibiting the expansion and activation of Th1 cells and Th17 cells and by promoting the development of M2 macrophages and regulatory T cells (Treg). Within a tumor microrenvironment, however, IL-10 can inhibit the expansion of both Treg and myeloid-derived suppressor cells (MDSC). IL-10 exerts protective effects including limiting tissue damage in arthritic inflammation and promoting muscle regeneration after injury, but it also contributes to the persistence of viral infections. IL-10 signals through a receptor complex composed of IL-10 R alpha and IL-10 R beta. IL-10 R beta additionally associates with IL-20 R alpha, IL-22 R alpha 1, or IL-28 R alpha to form the receptor complexes for IL-22, IL-26, IL-28, and IL-29.

397106

Q29055

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

1.Incubate IL-10 secreting cells in an antibody-coated well.
2.Remove cells by washing. Secreted IL-10 is captured by the immobilized antibody.
3.Incubate with biotinylated anti-IL-10 antibody.
4.Remove unbound biotinylated antibody by washing. Incubate with alkaline phosphatase conjugated streptavidin.
5.Wash to remove unbound enzyme.
6.Add substrate and monitor the formation of colored spots. Analyze using either an ELISpot reader or dissection microscope.