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
Greimers, Roland; Trebak, M.; Moutschen, Michelet al.
[en] 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).
Disciplines :
Immunology & infectious disease Biotechnology
Author, co-author :
Greimers, Roland ; Université de Liège - ULiège > Département des sciences biomédicales et précliniques > Anatomie et cytologie pathologiques
Trebak, M.
Moutschen, Michel ; Université de Liège - ULiège > Département des sciences cliniques > Immunopathologie - Transplantation
Jacobs, Nathalie ; Université de Liège - ULiège > Département des sciences biomédicales et précliniques > Anatomie et cytologie pathologiques
Boniver, Jacques ; Université de Liège - ULiège > Département des sciences biomédicales et précliniques > Anatomie et cytologie pathologiques
Language :
English
Title :
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
Publication date :
01 March 1996
Journal title :
Cytometry
ISSN :
0196-4763
eISSN :
1097-0320
Publisher :
John Wiley & Sons, Hoboken, United States - New Jersey
Volume :
23
Issue :
3
Pages :
205-17
Peer reviewed :
Peer Reviewed verified by ORBi
Funders :
F.R.S.-FNRS - Fonds de la Recherche Scientifique [BE]
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