Human IFN-gamma/IL-10 Dual-Color ELISpot Kit (CAT#: ITS-0322-P159)

1 Kit

1.Human IFN-gamma/IL-10 Microplate.
2.Biotin-conjugated Detection Antibody.
3.HRP-conjugated Detection Antibody.
4.Streptavidin-conjugated to Alkaline Phosphatase.
5.Dilution Buffers.
6.Wash Buffer Concentrate.
7.BCIP/NBT Chromogen.
8.AEC Chromogen.
9.Human IFN-gamma/IL-10 Positive Controls.

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 35 mins to 4 hours 50 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.Positive Controls - Reconstitute the lyophilized Human IFN-γ Positive Control and Human IL-10 Positive Control with 250 μL of culture medium that is used to incubate cells.
3.Detection Antibody Mixture (Concentrate A + Concentrate B) - Tap or vortex each vial to release reagent collected in the cap. Transfer 100μL of Detection Antibody Concentrate A and 200 μL of Detection Antibody Concentrate B into the vial labeled Dilution Buffer 1 and mix well.
4.Streptavidin-AP Concentrate A - Tap or vortex 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.
5.AEC Chromogen Solution - Transfer 250μL of AEC Chromogen to the vial labeled AEC Chromogen Buffer 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.
4.Aspirate each well and wash, repeating the process three times for a total of four washes.
5.Add 100μL 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 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.
11.Add 100 μL of AEC Chromogen Solution into each well, and incubate for 20 minutes at room temperature. Protect from light.
12.Decant the AEC Chromogen Solution 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 microplate thoroughly with paper towels, and dry completely either at room temperature (60-90 minutes) or 37°C (15-30 minutes).

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.
AEC Chromogen may cause skin, eye, and respiratory irritation. Avoid breathing fumes.
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 product at 2 - 8 °C.

Do not use past kit expiration date. This kit is validated for single use only.

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.

1.Two different proteins secreted by cultured cells bind to the capture antibodies coated on the well.
2.Remove cells by washing. Add the biotinylated detection antibody which binds to the FIRST captured protein and the HRP-conjugated detection antibody which binds to the SECOND captured protein.
3.Wash to remove any unbound antibodies.
4.Add the alkaline phosphatase-conjugated streptavidin
5.Wash to remove unbound enzyme.
6.Add BCIP/NBT Substrate to develop the blue spots representing the FIRST protein. After finishing incubation with the BCIP/NBT Substrate, wash the wells with deionized water and add AEC Chromogen to develop the red spots representing SECOND protein. Analyze using either an ELISpot reader capable of detectiong blue and red spots or a dissection mircoscope.