ELISpot and FluoroSpot for T Cell Response and Potency by PhDs at 20+ Year, FDA-Inspected Lab

ELISpot puts one visible spot on a membrane for every single cell that secretes the analyte you are measuring, so instead of an average concentration you get a count: how many of your cells actually respond to the antigen. That per-cell frequency is why vaccine and immunotherapy programs use it to measure antigen-specific T cell response, at sensitivities down to one responding cell in several hundred thousand.

FluoroSpot is ELISpot with fluorescent detection, which lets up to three analytes be read from the same single cell. Where ELISpot tells you how many cells respond, FluoroSpot tells you how many respond polyfunctionally, IFN-gamma with IL-2 and TNF-alpha from one cell, which is often the more meaningful measure of T cell quality in vaccine and immunotherapy work.

They answer neighboring questions. ELISpot counts secreting cells with exceptional sensitivity and modest cell numbers, and qualifies well for regulated use. ICS by flow phenotypes the responding cells, which subsets, which markers, at the cost of more cells and complexity. Programs often run ELISpot as the workhorse frequency readout and reserve flow for when the phenotype matters. Both run here, so the choice follows the question, not the equipment.

It matters enormously, and it is a logistics decision as much as a science one. Cryopreserved PBMC make multi-site trials feasible, but only if isolation, freeze and thaw are controlled; a mishandled thaw kills the very cells you are counting. PBMC are isolated here within 8 to 24 hours of draw, frozen at controlled rate, and thawed directly into the assay, with viability QC recorded before any well is counted.

In writing, before the study runs. Positive call rules, background subtraction and plate acceptance criteria are agreed during qualification, not improvised at readout, because a response definition invented after the data exists convinces no reviewer. Plates are counted on a CTL ImmunoSpot S6 Ultra M2 reader with the counting parameters locked and documented.

As a cell-frequency readout, yes, when the assay is qualified for its context of use: precision, linearity, robustness and analyst-to-analyst agreement are all established, GCLP for clinical work. And because supernatants from the same stimulation can be read by MSD or Luminex, one draw yields both the frequency of responding cells and the concentration of what they secreted.

Yes. Splenocyte and PBMC ELISpot from rodent and non-rodent tox or efficacy studies runs here alongside the clinical formats, which lets a vaccine or cell therapy program carry one readout logic from mouse to first-in-human instead of changing rulers mid-program.

Killing, measured cell by cell. Granzyme B release is a functional cytotoxicity readout for CAR-T, TCR-T and NK cell products, used in characterization and potency contexts where the question is not whether cells are present but whether they still kill. It slots beside proliferation and cytokine readouts to make a potency panel.