Hi Vanessa,

Make sure these are actual cells in the medium only well. Sometime yeast (grape like structure) also grows. It happened to me once, when I saw more cells in the experimental plate than the control plate. These cells were turned out to be yeast cells (grape like structure). Yeast infection is a common thing with the medium especially when you warm your medium in 37C water bath.

Best,

P. Ramaraj

Hi Vanessa,

I would believe your media got contaminated with bacterial components, such as LPS. Depending on the immunological status of your cells previous to the stimulus, you may detect more spots than expected with these type of contaminants, even in control conditions.

Another interesting point to pay attention is the spot size among different stimuli. Are the control well spots similar in size with those detected in the presence of your specific antigen? Sometimes, different stimulation may generate spots of different size and intensity.

Thus, I suggest you to add antibiotics to your media previous to its addition to the cells on the ELISPOT plate.

Best of luck,

Eduardo