ELISpot

ELISpot counts the cells secreting a cytokine - each spot is one responding cell. The raw counts only become comparable once you subtract background and normalize to the number of cells plated. Gradiance does both and reports spot-forming units in the standard units.

What it measures

The frequency of antigen-specific responding cells, expressed as spot-forming units (SFU) per a fixed number of input cells - typically per 10⁶ PBMC. The antigen-stimulated count minus the unstimulated background is the signal.

Data you upload

• Spot counts per well, with stimulated and unstimulated (background) conditions identified.
• The number of cells plated per well so counts can be normalized.
• Optional grouping (donor, timepoint, antigen) for comparison.

What you get back

• Background-subtracted SFU normalized per 10⁶ cells, per condition.
• A positivity call against the background using a standard response-definition rule.
• A comparison plot across groups or timepoints, with the relevant significance test.

Method note

Counts are first averaged across replicate wells, then the mean background is subtracted before scaling to the per-million-cells basis. Positivity uses a distribution-free rule (stimulated exceeding background by both a fold and an absolute margin) rather than a bare t-test, which is unreliable at the low spot counts ELISpot often produces. Group comparisons hand off to the same machinery as Group Comparison.

Related

For soluble cytokine quantification, see Multiplex Panels. For flow cytometry population frequencies, see Flow Summary.