IMPROVED 4-COLOR FLOW-CYTOMETRY METHOD USING FLUO-3 AND TRIPLE IMMUNOFLUORESCENCE FOR ANALYSIS OF INTRACELLULAR CALCIUM-ION ([CA2-NODE B-LYMPHOCYTE AND T-LYMPHOCYTE SUBSETS(]I) FLUXES AMONG MOUSE LYMPH)

A visible-light, dual-laser, now cytometric method was developed for t he simultaneous analysis of intracellular ionized calcium concentratio n ([Ca2+](i)) and three cell-surface markers (CD4, CD8, and Thy-1.2 an tigens) by using the calcium probe fluo-3 and using R-phycoerythrin (P E), peridinin chlorophyll-alpha protein (PerCP), and allophycocyanin ( APC) conjugated monoclonal antibodies (MoAbs), This improved method wa s used in the analysis of [Ca2+](i) mobilization upon in vitro stimula tion with mitogenic lectins [phytohaemagglutinin (PHA) or concanavalin A (ConA)], anti-CD3 MoAbs, or A23187 calcium ionophore in the heterog eneous lymph node cell populations from healthy C57BL/Ka mice, The pre sent results show that the calcium responses were heterogeneous and de pendent on the cellular immunophenotype, not only on lectins or anti-C D3 MoAbs stimulation, but also on the receptor-independent A23187 iono phore stimulation, An in situ fluo-3 calibration method (using A23187 and metabolic poisons in Ca2+/EGTA buffers with known free calcium con centrations) indicated a resting [Ca2+](i) in lymphocytes of 103 +/- 2 3 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 subset s showed an increase of fluo-3 fluorescence upon in vitro stimulation with ConA or PHA, but the calcium mobilization following lectin stimul ation was time delayed in all T-cell subsets, Only the T cells, includ ing the CD4(+)Thy-1(-) subset, responded to anti-CD3 MoAbs. The percen tage of responding cells upon stimulation with ConA was higher in T ce lls than in B cells, By contrast, PHA gave a higher response in B cell s. After stimulation with different mitogens, [Ca2+](i) increased in b oth CD4(+) and CD8(+) T-cell subsets, However, the percentage of respo nding cells was far higher in the CD4(+)Thy-1(+) subset than in the CD 4(+)Thy-1(-) or the CD8+ T-cell subsets, The stimulation with A23187 i onophore induced a higher calcium response in B cells than in T cells, Interestingly, it also induced greater Ca2+ mobilization in CD4(+) th an in CD8+ T cells, These results demonstrate the potential use of flu o-3 simultaneously with three fluorescein (FITC)-compatible fluorochro mes, This technique may be useful for investigating the role of the CD 4(+)Thy-1(-) T cells, a rare subset that is abnormally expanded in a m urine acquired immunodeficiency syndrome (murine AIDS). (C) 1996 Wiley -Liss, Inc.