Automated analysis of two- and three-color fluorescent Elispot (Fluorospot) assays for cytokine secretion

Abstract

The Elispot effectively measures the frequencies of cells secreting particular molecules, especially low-frequency cells such as antigen-specific T cells. The Fluorospot assay adapted this analysis to two products per cell, and this has now been extended to three-color measurement of both mouse and human cytokine-secreting cells. Due to the increased data complexity, and particularly the need to define single-, double- and triple-producing cells, it is critical to objectively quantify spot number, size, intensity, and coincidence with other spots. An automated counting program, Exploraspot, was therefore developed to detect and quantify Fluorospots in automated fluorescence microscope images. Morphological parameters, including size, intensity, location, circularity and others are calculated for each spot, exported in FCS format, and further analyzed by gating and graphical display in popular flow cytometry analysis programs. The utility of Exploraspot is demonstrated by identification of single-, double- and triple-secreting T cells; tolerance of variable background fluorescence; and estimation of the numbers of genuine versus random multiple events.

Figure 1. Two- and three-color Fluorospot assays detect various cell phenotypes

A) 2×10³ Th1 cells per well were restimulated in a Fluorospot assay for MIP-1α (red, Cy3) and IFN-γ (green, Cy2). B) 2×10³ Th1 and 7×10³ Th2 cells per well were restimulated in a Fluorospot assay for IL-4 (red, Cy3) and IFN-γ (green, Cy2). C) CD4 T cells expressing a range of cytokine secretion phenotypes were restimulated in a Fluorospot assay for IL-5 (red, Cy3), IL-2 (green, Cy2) and IFN-γ (blue, Cy5). The top three rows show single-channel images for red, green and blue, and the bottom rows show the composite images.

Figure 2. Exploraspot processing routine for a three-color Fluorospot experiment

Figure 3. Assignment and analysis of coincidence limits using matched and mismatched images

Images were used from the experiments shown in Figure 1C (A) or Fig. 1A (B, C, D, E). A) Assignment of a coincidence limit (CL) for two pairs of spots. At CL=5 pixels, the green and blue spots would be considered as one (left) or two (right) events. B) Effect of matching and mismatching the red and green channels of duplicate wells in the two-color MIP-1α and IFNγ assay (most are genuine double-producers). C) The number of apparent double-producers found at different values of the coincidence limit, using the matched and mismatched wells shown in B. D) The frequency of random doubles with increasing numbers of spots per well, estimated from mismatched images. E) The coincidence curves comparing ratios for ‘predicted random’ to actual matched and mismatched images.

Figure 4. IFNγ and MIP-1α intensity time-course

Exploraspot intensity histograms of IFN-γ (left) and MIP-1α (right) production over time (converted to FCS format and visualized using FlowJo). Data pooled from 6 replicate wells, each containing 1×10³ Th1 cells per well.

Figure 5. Quadrant plots of two-color spot intensities for two experiments

Exploraspot dual intensity plots of A) MIP-1α and IFN-γ production by 8×10³ Th1 cells (pooled from 4 replicates), and B) IL-4 and IFN-γ production of a mixture of 8×10³ Th1 and 2.8×10⁴ Th2 cells (pooled from 4 replicates).

Figure 6. Examples of Normal, Low, and High Background

A) Normal image: Image data from a Th1 IFNγ Fluorospot plate. B) Low Background image: Image data from a negative control well from same Th1 IFN-γ Fluorospot plate. C) High Background image: Image data from a separate Th1 IFNγ experiment with high background and low spot count. For display purposes, all three original images have been enhanced by equal brightness adjustments, but all calculations were performed on the original images. D) Fidelity of spot-finding in the presence of increased background.