No Spots or Very Weak Signal

Symptom

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.

1. 1 Cells not incubated long enough or insufficient time to respond to stimulant
2. 2 Cells not stimulated correctly or inappropriate stimulation conditions
3. 3 Inadequate color development due to improper reagent storage or loss of activity
4. 4 Insufficient cells per well (below optimal 1-2 × 10⁵ cells range)
5. 5 Primary or secondary antibody concentration too low

1. 1 Increase cell incubation time or use indirect method (pre-treat cells with stimulant)
2. 2 Use positive stimulation control with known inducer of target cytokine/protein
3. 3 Monitor color development with overhead microscope; verify reagent storage and activity
4. 4 Increase cell number to 1-2 × 10⁵ cells per well; optimize as needed
5. 5 Increase primary and/or secondary antibody concentrations through optimization

Related Video (2)

Mabtech ★ 92

ELISpot tutorial: step-by-step full assay protocol

"Full step-by-step ELISpot protocol with tips directly addresses correct incubation timing and development steps that cause weak or absent spots."

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Elispot assay ( assay to determine cytokine producing cells)

"Overview of the ELISpot assay explains cell culture and cytokine secretion principles behind why insufficient incubation produces few spots."