The FluoroSpot assay is an inventive development of the ELISpot assay and uses fluorescent detection instead of colorimetric. This allows for simultaneous detection of several analytes, which is a major advantage. The human IFN-γ/Granzyme B/TNF-α FluoroSpot kit is perfect for users who wish to have a convenient assay with minimized assay variability. It enables enumeration of cells secreting one, two or three analytes. The kit includes low fluorescent plates pre-coated with monoclonal capture antibodies, monoclonal detection antibodies, secondary reagents conjugated to fluorophores, a fluorescence enhancer and an anti-CD28 monoclonal antibody for co-stimulation. A polyclonal activator for human T cells (mAb CD3-2) is supplied as a positive control for cytokine secretion.