Like an ELISpot assay, FluoroSpot is used to detect and analyse the secreted products of plated cells. However, instead of relying on a colorimetric readout, FluoroSpot uses fluorescently-labelled antibodies for detection. By measuring the intensity of the fluorescent readouts, the amount of each secreted product can be quantified.
Highly-sensitive and easy to perform, FluoroSpot can be used to analyse the secretion of multiple analytes from the same cell. It can also distinguish between different cell types in the same assay well and can inform the relative robustness of similar cell types within an assay by comparing spot sizes and fluorescent intensities. A further utility of FluoroSpot is to investigate the downstream effects of biomolecular co-secretion.
Analysis of FluoroSpot data requires a specialised reader, capable of measuring the volume of each spot and of detecting different wavelengths. Careful selection of fluorophores allows simultaneous analysis of multiple secreted molecules which may exhibit different kinetics, while fixed camera settings can eliminate user bias.