No Spots or Very Weak Signal (ELISPOT)

Question

Accepted Answer

Few or no spots are visible after development, or spots are extremely faint and difficult to detect even though cytokine-secreting cells should be present. Common causes: 1) Cells not incubated long enough or insufficient time to respond to stimulant 2) Cells not stimulated correctly or inappropriate stimulation conditions 3) Inadequate color development due to improper reagent storage or loss of activity 4) Insufficient cells per well (below optimal 1-2 × 10⁵ cells range) 5) Primary or secondary antibody concentration too low Solutions: 1) Increase cell incubation time or use indirect method (pre-treat cells with stimulant) 2) Use positive stimulation control with known inducer of target cytokine/protein 3) Monitor color development with overhead microscope; verify reagent storage and activity 4) Increase cell number to 1-2 × 10⁵ cells per well; optimize as needed 5) Increase primary and/or secondary antibody concentrations through optimization