The enzyme-linked immunosorbent spot (ELISpot) assay performe methods to measure antigen-specific T cells activation. ELISpot is highly quantitative, can measure a broad range of response magnitudes, and can assess critical cellular immune-related activities. In this assay, cells are cultured on a surface coated with a specific capture antibody in the presence or absence of stimuli. Proteins, such as cytokines, that are secreted by the cells will be captured by the specific antibodies on the surface. The ELISpot assay captures the presence of cytokines immediately after secretion and the high sensitivity of the assay makes it particularly useful for studies of the small population of cells found in specific immune responses. The ELISpot technique is not limited to measurement of cytokines; it is also suitable for almost any secreted protein where single-cell analysis is of interest.

PBMC were isolated from whole blood collected from two self-declared healthy donors and tested in presence of positive controls (PHA and LPS) to stimulate the lymphocytes T or media alone as negative control using enzymatic ELISPOT assay (colorimetric read out) and FluoroSpot assay (fluorescence read out). The assays were performed and the expression of cytokines was measured after 24h of PBMC stimulation and each condition was tested in triplicates.

The reading output was spot counts representing IFN-γ (red color) /IL-2 (blue color) secretion for the double-color enzymatic Elispot assay and IFN-γ (green) /IL-2 (yellow)/Granzyme B (red) secretion for the three-color fluorospot assay using the CTL ImmunoSpot S6 FluoroCore Analyzer (spot counts).