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

Cytometry. 1996 Mar 1;23(3):205-17. doi: 10.1002/(SICI)1097-0320(19960301)23:3<205::AID-CYTO4>3.0.CO;2-H.


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).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aniline Compounds / pharmacology*
  • Animals
  • B-Lymphocytes / drug effects
  • B-Lymphocytes / immunology
  • B-Lymphocytes / metabolism*
  • CD4-Positive T-Lymphocytes / drug effects
  • CD4-Positive T-Lymphocytes / immunology
  • CD4-Positive T-Lymphocytes / metabolism*
  • CD8-Positive T-Lymphocytes / drug effects*
  • CD8-Positive T-Lymphocytes / immunology
  • CD8-Positive T-Lymphocytes / metabolism*
  • Calcimycin / pharmacology
  • Calcium / metabolism*
  • Carotenoids / pharmacology
  • Flow Cytometry / methods*
  • Fluorescent Dyes / pharmacology*
  • Ionophores / pharmacology
  • Lectins / pharmacology
  • Lymph Nodes / immunology
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Mitogens / pharmacology
  • Phycocyanin / pharmacology
  • Phycoerythrin / pharmacology
  • Protozoan Proteins / pharmacology
  • Stimulation, Chemical
  • T-Lymphocytes / drug effects
  • T-Lymphocytes / immunology
  • T-Lymphocytes / metabolism
  • Thy-1 Antigens / immunology
  • Xanthenes / pharmacology*


  • Aniline Compounds
  • Fluorescent Dyes
  • Ionophores
  • Lectins
  • Mitogens
  • Protozoan Proteins
  • Thy-1 Antigens
  • Xanthenes
  • allophycocyanin
  • peridinin chlorophyll-a protein, Dinophyceae
  • Phycocyanin
  • Phycoerythrin
  • Fluo-3
  • Carotenoids
  • Calcimycin
  • Calcium