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.
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 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).
authors.affiliation: Laboratory of Pathological and Cytological Anatomy, University Hospital of Liege, Belgium.
authors.forename: R
authors.initials: R
authors.lastname: Greimers
authors.forename: M
authors.initials: M
authors.lastname: Trebak
authors.forename: M
authors.initials: M
authors.lastname: Moutschen
authors.forename: N
authors.initials: N
authors.lastname: Jacobs
authors.forename: J
authors.initials: J
authors.lastname: Boniver
mesh_terms: D000814:Aniline Compounds / Q000494:pharmacology*; D000818:Animals; D001402:B-Lymphocytes / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D015496:CD4-Positive T-Lymphocytes / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D018414:CD8-Positive T-Lymphocytes / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism*; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002338:Carotenoids / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000494:pharmacology*; D007476:Ionophores / Q000494:pharmacology; D037102:Lectins / Q000494:pharmacology; D008198:Lymph Nodes / Q000276:immunology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008934:Mitogens / Q000494:pharmacology; D010798:Phycocyanin / Q000494:pharmacology; D010799:Phycoerythrin / Q000494:pharmacology; D015800:Protozoan Proteins / Q000494:pharmacology; D013268:Stimulation, Chemical; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D018800:Thy-1 Antigens / Q000276:immunology; D014966:Xanthenes / Q000494:pharmacology*
chemical_list: D000814:Aniline Compounds; D005456:Fluorescent Dyes; D007476:Ionophores; D037102:Lectins; D008934:Mitogens; D015800:Protozoan Proteins; D018800:Thy-1 Antigens; D014966:Xanthenes; C002497:allophycocyanin; C073227:peridinin chlorophyll-a protein, Dinophyceae; D010798:Phycocyanin; D010799:Phycoerythrin; C059715:Fluo-3; D002338:Carotenoids; D000001:Calcimycin; D002118:Calcium