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, Mohamed; Moutschen, Michel; Jacobs, Nathalie; Boniver, Jacques

doi:10.1002/(sici)1097-0320(19960301)23:3<205::aid-cyto4>3.0.co;2-h

Cited by 38 publications

(25 citation statements)

References 45 publications

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“…However, flow cytometry and its multiparameter analysis allow additional characterization of the cells involved in the chemokine receptor-mediated signal transduction. Cytometry Part A 51A: [35][36][37][38][39][40][41][42][43][44][45]2003. © 2002 Wiley-Liss, Inc.…”

Section: Discussionmentioning

confidence: 99%

“…Several studies have found that Fluo-3 is a valuable dye for flow cytometric analysis of intracellular Ca 2ϩ changes in various cell types (32)(33)(34)(35)(36)(37)(38)(39).…”

Section: Discussionmentioning

confidence: 99%

“…Previous studies by other groups have proved that this assay has interesting advantages over the "cuvet" method because measurement of Ca 2ϩ responses can be correlated with the determination of several other cell parameters (32)(33)(34)(35)(36)(37)(38)(39). We used both methods to compare Ca 2ϩ signaling in five different CXCR4-expressing leukemic cell lines and in PBMCs.…”

Section: Discussionmentioning

confidence: 99%

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Evaluation of SDF‐1/CXCR4‐induced Ca²⁺ signaling by fluorometric imaging plate reader (FLIPR) and flow cytometry

et al. 2002

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BackgroundThe chemokine receptors CXCR4 and CCR5 are the main coreceptors for human immunodeficiency virus (HIV) 1 to enter its target cells. The antiviral activity of their natural ligands (stromal cell‐derived factor 1 [SDF‐1], regulated on activation normal T‐cell expressed and secreted, (RANTES) and macrophage inflammatory proteins 1α and 1β, MIP‐1α and MIP‐1β) and the finding that individuals deficient in CCR5 are relatively resistant to HIV infection led to the concept that chemokine receptor antagonists can play an important role in anti‐HIV therapy. AMD3100, the prototype compound of the bicyclams, is one of the most potent and selective CXCR4 antagonists described to date. The search for new chemokine receptor antagonists involves the evaluation of compounds for their ability to block the specific chemokine‐induced transient intracellular Ca2+ flux. We evaluated two cell‐based‐fluorescent methods with the use of the Fluorometric Imaging Plate Reader (FLIPR) system and a flow cytometric assay to measure the SDF‐1–induced intracellular Ca2+ mobilization via CXCR4. Both assay systems were compared for their sensitivity, advantages, and system‐dependent limitations.MethodsCXCR4+ lymphocytic and monocytic cell lines commonly used in laboratories studying acquired immunodeficiency syndrome and freshly isolated peripheral blood mononuclear cells (PBMCs) were loaded with the Ca2+ indicator Fluo‐3. After washing, the cells were preincubated with the CXCR4 antagonist AMD3100. Then the increase in intracellular Ca2+ concentration after stimulation with SDF‐1 was examined by the FLIPR and the flow cytometer, which monitored the change in green fluorescence intensity of Ca2+‐bound Fluo‐3. Surface CXCR4 expression was also determined flow cytometrically.ResultsIn all five CXCR4+ cell lines, SDF‐1 elicited a transient increase in intracellular Ca2+ concentration. For each cell line, the magnitude of response was related to the level of CXCR4 expression: the cells with the highest CXCR4 level showed the strongest Ca2+ response. AMD3100 effected a dose‐dependent inhibition of the SDF‐1–induced Ca2+ flux in all cell lines examined. The FLIPR was more sensitive than the flow cytometer in detecting minor Ca2+ responses. In freshly isolated PBMCs, the Ca2+ response was due solely to the stimulation of monocytes and granulocytes, whereas the lymphocyte population, although expressing CXCR4, did not respond at all. This phenomenon could be observed with the flow cytometer and not with the FLIPR.ConclusionsThe FLIPR system is a most adequate to monitor intracellular Ca2+ mobilization and to evaluate chemokine receptor antagonists. However, flow cytometry and its multiparameter analysis allow additional characterization of the cells involved in the chemokine receptor‐mediated signal transduction. Cytometry Part A 51A:35–45, 2003. © 2002 Wiley‐Liss, Inc.

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Section: Discussionmentioning

confidence: 99%

“…Several studies have found that Fluo-3 is a valuable dye for flow cytometric analysis of intracellular Ca 2ϩ changes in various cell types (32)(33)(34)(35)(36)(37)(38)(39).…”

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Evaluation of SDF‐1/CXCR4‐induced Ca²⁺ signaling by fluorometric imaging plate reader (FLIPR) and flow cytometry

et al. 2002

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“…Cells (1 ϫ 10 7 per ml) were incubated in PBS with 2 M fluo-3 AM and 0.02% pluronic acid F-127 (Molecular Probes) for 20 min at room temperature. Next, the cells were diluted to a final concentration of 2 ϫ 10 6 per ml with PBS containing 1% FCS, and incubated for an additional 40 min in a 37.5 Ϯ 0.1°C water bath (16). Cells were then washed twice with PBS and maintained in RPMI medium 1640 (Cellgro), buffered with 25 mM Hepes and supplemented with 10% FBS͞2 mM L-glutamine (Cellgro).…”

Section: Methodsmentioning

confidence: 99%

Presentation of antagonist peptides to naive CD4⁺T cells abrogates spatial reorganization of class II MHC peptide complexes on the surface of dendritic cells

Chmielowski

Pacholczyk

Kraj

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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A ntigenic peptides with affinity sufficient to activate T cell response, so-called agonists, are not the only peptides encountered by the T cell receptor (TCR) on the surface of antigen-presenting cells (APCs). Usually, TCR faces a myriad of different peptides bound to MHC molecules, which are detained in the interface between the APC and the T cell, most of which have undetectable (i.e., null) affinity for the TCR. The rules and mechanisms that govern the trafficking of MHC͞peptide complexes on the membrane of the APC, which are involved in the regulation of the T cell response, are not well understood. Some recent evidence suggests that APCs can autonomously organize the distribution of MHC͞peptide complexes on their surface. Colocalization of a diverse set of these complexes within lipid rafts may improve the presentation of less frequent peptides to T cells, whereas the accumulation of class II MHC molecules bound with a few abundant peptides in tetraspan microdomains may be a favorable target for particular subpopulations of T cells (1, 2). On the other hand, the qualitative sorting of MHC͞ peptide complexes on APCs is also actively driven by TCRs during the onset of T cell activation (3). Initially, it was reported that on activation, CD4 ϩ T cells selectively cluster agonist class II MHC͞peptide complexes, but in subsequent studies it was found that null class II MHC͞peptide complexes with weak or no affinity for TCRs are also present within the cell-cell contact site (3, 4). It was also shown that TCR engagement by MHC molecules loaded with so-called antagonist peptide may inhibit T cell response (5). It is not clear whether this effect is because of the direct interference with TCR binding to agonist MHC͞ peptide complexes (6-8) or because of the interference with the formation of functional MHC͞peptide clusters at the T cell-APC interface, as observed for other receptor͞ligand interactions (9). To investigate the latter possibility we analyzed the effect of exogenously added antagonist peptide on the redistribution of agonist and null class II MHC͞peptide complexes in the interface area of dendritic cells (DCs) interacting with naive CD4 ϩ T cells expressing transgenic antigen-specific TCR (TCR Tg ). Our results demonstrate that in contrast to the interaction with effector CD4 ϩ T cells, interaction with naive CD4 ϩ T cells results in expulsion of null MHC͞peptide complexes from the T cell-DC interface and that this process in inhibited in the presence of antagonist peptides. Materials and MethodsMice. Mice deficient for invariant chain (A b Ii Ϫ ) were kindly provided by R. Germain (National Institutes of Health, Bethesda). Mice transgenic for the V␣4JV␤8.1 TCR (TCR Tg ) were generated at the Medical College of Georgia (Augusta) (10). All mice were housed under specific pathogen-free conditions in the animal care facility at the Medical College of Georgia. DNA encoding CD3 (kindly provided by Makio Iwashima, Medical College of Georgia) was cloned into pECFP-N1 and pEYFP-N1 vectors. DNA fragments enco...

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“…Fluo-3 AM loading of purified B cells and surface immunostaining of unfractionated splenocytes was performed using the method described before, with minor modifications (43). Cells were resuspended at 2 ϫ 10 6 /ml in HBSS with 1 M fluo-3-acetoxymethyl ester (fluo-3 AM) and 0.5 g/ml pluronic F-127 (Molecular Probes) and incubated for 30 min at 37°C.…”

Section: Ca 2ϩ Influx Measurementsmentioning

confidence: 99%

Antinuclear Antigen B Cells That Down-Regulate Surface B Cell Receptor during Development to Mature, Follicular Phenotype Do Not Display Features of Anergy In Vitro

Liu

Manser

2005

The Journal of Immunology

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We previously demonstrated that B cells expressing a transgenic BCR with “dual reactivity” for the hapten arsonate and nuclear autoantigens efficiently complete development to follicular phenotype and stably reside in follicles in vivo. These B cells express very low levels of surface IgM and IgD, suggesting that they avoid central deletion and peripheral anergy by reducing their avidity for autoantigen via surface BCR (sBCR) down-regulation. Since a variety of states of B cell anergy have been previously described, a thorough examination of the functional capabilities of these B cells was required to test this hypothesis. In this study, we show that surface Ig cross-linking induces amounts of proximal BCR signaling in these B cells commensurate with their reduced sBCR levels. Functionally, however, they are comparable to nonautoreactive B cells in cell cycle progression, up-regulation of activation and costimulatory molecules, and Ab-forming cell differentiation when treated with a variety of stimuli in vitro. In addition, these B cells can efficiently process and present Ag and are capable of undergoing cognate interaction with naive TCR-transgenic T cells, resulting in robust IL-2 production. Together, these data reveal a lack of intrinsic anergy involving any known mechanism, supporting the idea that this type of antinuclear Ag B cell becomes indifferent to cognate autoantigen by down-regulating sBCR.

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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

Cited by 38 publications

References 45 publications

Evaluation of SDF‐1/CXCR4‐induced Ca²⁺ signaling by fluorometric imaging plate reader (FLIPR) and flow cytometry

Evaluation of SDF‐1/CXCR4‐induced Ca²⁺ signaling by fluorometric imaging plate reader (FLIPR) and flow cytometry

Presentation of antagonist peptides to naive CD4⁺T cells abrogates spatial reorganization of class II MHC peptide complexes on the surface of dendritic cells

Antinuclear Antigen B Cells That Down-Regulate Surface B Cell Receptor during Development to Mature, Follicular Phenotype Do Not Display Features of Anergy In Vitro

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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

Cited by 38 publications

References 45 publications

Evaluation of SDF‐1/CXCR4‐induced Ca2+ signaling by fluorometric imaging plate reader (FLIPR) and flow cytometry

Evaluation of SDF‐1/CXCR4‐induced Ca2+ signaling by fluorometric imaging plate reader (FLIPR) and flow cytometry

Presentation of antagonist peptides to naive CD4+T cells abrogates spatial reorganization of class II MHC peptide complexes on the surface of dendritic cells

Antinuclear Antigen B Cells That Down-Regulate Surface B Cell Receptor during Development to Mature, Follicular Phenotype Do Not Display Features of Anergy In Vitro

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Evaluation of SDF‐1/CXCR4‐induced Ca²⁺ signaling by fluorometric imaging plate reader (FLIPR) and flow cytometry

Evaluation of SDF‐1/CXCR4‐induced Ca²⁺ signaling by fluorometric imaging plate reader (FLIPR) and flow cytometry

Presentation of antagonist peptides to naive CD4⁺T cells abrogates spatial reorganization of class II MHC peptide complexes on the surface of dendritic cells