What are the characteristics of a spot?
Each spot within an ELISPOT well is the ‘footprint’ of a single cell that has released a relatively high amount of cytokines. True spots have a dense center with a light outer ring caused by the diffusion of the cytokine from the producing cell. The color depth or the size of spots depends on the amount of secreted cytokines. Only strong and well-defined spots should be counted; any small or faint spot is likely to be an artifact and should be ignored. Artifactual spots can be caused by the aggregation of antibodies used in the process of coating and detection. Also incomplete removal of cells from the plate after incubation can be a source for artifactual spots. A simple measure to minimize false spot formation is to thoroughly wash the plate between all incubation steps and after completion of the coloring reaction. Sometimes small and very dark spots appear in the background that are not generated by secreted cytokines. In the visual evaluation, these spots are differentiated from ‘
