Improved 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

Roland Greimers, Mohamed Trebak, Michel Moutschen, Nathalie Jacobs, Jacques Boniver

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33 Scopus citations

Abstract

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 Thy1.2 antigens) by using the calcium probe fluo-3 and using R-phycoerythrin (PE), peridinin chlorophyll-α protein (PerCP), and allophycocyanin (APC) conjugated monoclonal antibodies (MoAbs). This improved method was used in the analysis of [Ca3+]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).

Original languageEnglish (US)
Pages (from-to)205-217
Number of pages13
JournalCytometry
Volume23
Issue number3
DOIs
StatePublished - Mar 1 1996

All Science Journal Classification (ASJC) codes

  • Pathology and Forensic Medicine
  • Biophysics
  • Hematology
  • Endocrinology
  • Cell Biology

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