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No spots ELISPOT

Find solutions to no spots in ELISPOT.

Cells not incubated for long enough or may take time to respond to stimulant

Possible causes

Solution

  • Increase the cell incubation time or use indirect method (pre-treat cells with stimulant).

Ensure the cells are stimulated correctly

Possible causes

Solution

  • Use a positive stimulation control – a stimulant that you know will induce expression of your cytokine/protein of interest.

Inadequate color development

Possible causes

Solution

  • Monitor color development with an overhead microscope and ensure that the developing reagents have been stored correctly and have not lost activity.

Not enough cells secreting cytokine/protein of interest

Possible causes

Solution

  • Increase the number of cells. Cell numbers should usually range between 1- 2 X 105 cells per well, and this may require some optimization.

Not enough primary or secondary antibody

Possible causes

Solution

  • Concentration of the primary and/or secondary antibody will need to be increased. This will require optimization.