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See detailA Strategy for Multiple Immunophenotyping by Image Cytometry: Model Studies Using Latex Microbeads Labeled with Seven Streptavidin-Bound Fluorochromes
Gothot, André ULg; Grosdent, J. C.; Paulus, Jean-Michel ULg

in Cytometry (1996), 24(3), 214-25

Multiple immunophenotyping is aimed at identifying several cell populations in a single labeling procedure by their ability to bind combinations of specific labeled antibodies. The present work ... [more ▼]

Multiple immunophenotyping is aimed at identifying several cell populations in a single labeling procedure by their ability to bind combinations of specific labeled antibodies. The present work demonstrates the simultaneous discrimination by using image cytometry of aminomethylcoumarin acetate (AMCA), Lucifer yellow (LY), fluorescein isothiocyanate (FITC), R-phycoerythrin (PE), PE-Texas red tandem (Red613), peridinin-chlorophyll protein (PerCP), and allophycocyanin (APC), which were all bound to latex beads as streptavidin-conjugated fluorochromes. This has been the result of a step-by-step optimization of the several factors affecting the sensitivity and specificity of multiple immunofluorescence analysis. First, 14 streptavidin-conjugated fluorochromes were evaluated by using spectrofluorometry. A primary selection was then made of ten spectrally separable dyes that could be evaluated by using image cytometry. These dyes were bound to latex particles, and specific filter combinations were assembled to minimize crosstalk between fluorophores while preserving sufficient fluorescence intensity and counting statistics. Potential probe associations were then assessed by measuring the emissions of all fluorochromes that were detected by each filter combination. The resulting crosstalk matrix served as the basic tool both for final selection of the optimal filter combination and for dye set (composed, in this case, of the seven fluorochromes described above) and for mathematical correction of residual spectral overlap. Next, an image cytometry system was adapted to collect seven images of matched brightness with the selected combination of excitation/emission filters and dichroic mirrors. Finally, seven-parameter synthetic images were generated by digital image processing. [less ▲]

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See detailImproved Four-Color Flow Cytometry Method Using Fluo-3 and Triple Immunofluorescence for Analysis of Intracellular Calcium Ion ([Ca2+]I) Fluxes among Mouse Lymph Node B- and T-Lymphocyte Subsets
Greimers, Roland ULg; Trebak, M.; Moutschen, Michel ULg et al

in Cytometry (1996), 23(3), 205-17

A visible-light, dual-laser, flow cytometric method was developed for the simultaneous analysis of intracellular ionized calcium concentration ([Ca2+]i) and three cell-surface markers (CD4, CD8, and Thy-1 ... [more ▼]

A visible-light, dual-laser, flow cytometric method was developed for the simultaneous analysis of intracellular ionized calcium concentration ([Ca2+]i) and three cell-surface markers (CD4, CD8, and Thy-1.2 antigens) by using the calcium probe fluo-3 and using R-phycoerythrin (PE), peridinin chlorophyll-alpha protein (PerCP), and allophycocyanin (APC) conjugated monoclonal antibodies (MoAbs). This improved method was used in the analysis of [Ca2+]i mobilization upon in vitro stimulation with mitogenic lectins [phytohaemagglutinin (PHA) or concanavalin A (ConA)], anti-CD3 MoAbs, or A23187 calcium ionophore in the heterogeneous lymph node cell populations from healthy C57BL/Ka mice. The present results show that the calcium responses were heterogeneous and dependent on the cellular immunophenotype, not only on lectins or anti-CD3 MoAbs stimulation, but also on the receptor-independent A23187 ionophore stimulation. An in situ fluo-3 calibration method (using A23187 and metabolic poisons in Ca2+ /EGTA buffers with known free calcium concentrations) indicated a resting [Ca2+]i in lymphocytes of 103 +/- 23 nM (mean +/- S.D.) but with significant differences between the [Ca2+]i in B cells and in all of the T-cell subsets (CD4+Thy-1+, CD4+Thy-1-, and CD8+T cells). Both the B cells and the T-cell subsets showed an increase of fluo-3 fluorescence upon in vitro stimulation with ConA or PHA, but the calcium mobilization following lectin stimulation was time delayed in all T-cell subsets. Only the T cells, including the CD4+Thy-1- subset, responded to anti-CD3 MoAbs. The percentage of responding cells upon stimulation with ConA was higher in T cells than in B cells. By contrast, PHA gave a higher response in B cells. After stimulation with different mitogens, [Ca2+]i increased in both CD4+ and CD8+ T-cell subsets. However, the percentage of responding cells was far higher in the CD4+Thy-1+ subset than in the CD4+Thy-1- or the CD8+T-cell subsets. The stimulation with A23187 ionophore induced a higher calcium response in B cells than in T cells. Interestingly, it also induced greater Ca2+ mobilization in CD4+ than in CD8+T cells. These results demonstrate the potential use of fluo-3 simultaneously with three fluorescein (FITC)-compatible fluorochromes. This technique may be useful for investigating the role of the CD4+Thy-1-T cells, a rare subset that is abnormally expanded in a murine acquired immunodeficiency syndrome (murine AIDS). [less ▲]

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