What are the small, dark spots that are not generated by secreted cytokines?
There are two possibilities. Due tot static electricity, dust particles attach to the underside and/or inside of the well of the transparent polystyrene-bottomed plates. Upon visual evaluation, these spots vary in size and are highly irregular. By cleaning the underside of the well with 70% ethanol, followed by blowing 4-5 bar compressed air against the underside and into the well, this problem can easily be solved. The second possibility are cell wall fragments containing receptor-bound cytokines that stick to the coating antibodies in the well. These particles are very small, have sharp edges and cannot easily be removed by standard washing procedures. Remarkably, only PBMCs from certain individuals generate these ‘sticky’ spots. By proper gating, these small artifactual spots are ignored by standard ELISPOT readers.
