I did my first ELISpot using methods and reagents that yielded previously successful results. Detecting IFN-gamma by plating 1:1 ratio of splenocytes:draining lymph node cells.
See image. Wells D9 and H9 are my cocktail stimulated cells. These wells are the best results on my plate, and the spots are too dim to reliably detect on a plate reader.
As you can see in the picture, all other wells have no spots but have this weird coral pattern in the middle of the well. Has anyone encountered a similar coral pattern/artifact? What step in the ELISpot is likely to be causing this artifact?
I will do my best. Is there astep on the ELISPOT similar to precipitation?
I do agree it might be the cells. Maybe add the proteins...
Siqi Liu I wasn't able to see my cells under the media, so am unsure if that is the factor at play here.
Mª Angeles Zorrilla Lopez-Perea No precipitation-like step in ELISpot.
I repeated the assay protocol using BD antibodies instead of MABTECH and I have good spots now!
they can be collagen as they are sticky and together, you can add filters before adding cells to the plate or same time using collagenase