ELISPOT for IFN-gamma and IL-10

Wednesday, October 29, 2003

Description
An ELIspot assay to count cytokine-producting cells

Procedure
- Coat plates with primary Antibody 2 g/ml, 50 l/well
- Incubate overnight 4C
- Aspirate (do not remove the cover to keep the plate sterile)
- Incubate with antigen and cells (vol. 200 l) overnight 37C
- Wash 3 times like ELISA + 2 washings by aspiration
- Add secondary antibody (biotinylated) 1 g/ml, 100 l/well
- Incubate 45 minutes room Temp.
- Wash 3 times (aspiration)
- Add Avidin-peroxydase 1 g/ml, 100 l/well
- Incubate 30 minutes room Temp.
- Wash 5 times (aspiration)
- Add substrate 41CN (sigma), allow coloration to develop
- Wash 5 times with distilled water (aspiration)
- Count spots

Recipes

Supplies
For this assay, the Abs and plates were obtained from R&D systems

Tips

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ELISPOT for IFN-gamma and IL-10