Example of a Fluorospot reading

FluoroSpot analysis of human IFN-γ/IL-2 secreting cells. Peripheral blood mononuclear cells (PBMC; 200 000/well) were stimulated, in the presence of anti-CD28 antibody, for 16 hours with a pool of MHC class I-restricted peptides13 derived from CMV, EBV and Flu (available from Mabtech as “CEF peptide pool”).

Cells secreting both cytokines (about 40% of responding cells) create yellow spots in an overlay of green and red images. In the CD8+ T-cell mediated response to CEF, the majority of IL-2 secreting cells also produce IFN-γ. The number of spontaneous spots by unstimulated control cells were <1 in IFN-γ/IL-2 double staining. Wells were analyzed with an iSpot reader(AID GmbH).