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)

Animated biology With arpan ★ 82

Elispot assay ( assay to determine cytokine producing cells)

"Directly explains the ELISpot assay's dependence on cell culture time for cytokine detection, addressing the underlying cause of weak signal."

Mabtech ★ 75

ELISpot tutorial: step-by-step full assay protocol

"Step-by-step protocol includes incubation timing details, helping avoid insufficient stimulation periods that lead to no spots."