Skip to main content

Confluent spots in ELISPOT

Find solutions for confluent spots in ELISPOT.

Cells over-stimulated

Possible causes

Solution

  • Over-stimulation will result in a lot of cytokine/protein being secreted by the cell. This will produce spots that will start to merge and become indistinguishable. Reduce the amount of stimulant in the culture media or culture for a shorter amount of time.

Poor coating, too much antibody

Possible causes

Solution

  • Reduce primary antibody concentration.

Prolonged cell culture

Possible causes

Solution

  • The longer the cells are incubated, the more cytokine/protein they will secrete. This will result in larger spots that will start to merge and become indistinguishable. Reduce cell culture step incubation time. (It is generally advised not to exceed 24 hours).