Measurement of cytoplasmic calcium in lymphocytes using flow cytometry (original) (raw)
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Methods, 1991
dyes, such as quin-2, indo-1, and fluo-3, has greatly facilitated the study of this process. Indo-1 has been shown to be the calcium indicator of choice for measuring calcium responses by flow cytometry. The spectral properties of indo-1 allow measurement of [Ca2+], independent of heterogeneity in the cell uptake of dye. In this report methods for the determination of [Ca2+], responses in B lymphocytes using polyclonal stimulation by antiimmunoglobulin antibodies are described. In addition, it is shown that these techniques can be utilized for measuring responses in small subpopulations of cells. Specifically, as many as 4% of B cells obtained from donors previously vaccinated with pneumococcal polysaccharides can be induced to mobilize [Ca2+], after in vitro stimulation with purified polysaccharides. Because indo-1 has the disadvantage that it requires uv light for excitation, a fluorochrome that can be excited by visible light would be more generally applicable. Fluo-3 is a calcium-sensitive dye that can be excited by 488-nm illumination and can therefore be used on most argon-laser-based flow cytometers equipped with a fixed-filter setup for measurement of fluorescein and phycoerythrin (PE). In addition, fluo-3 can be combined with PE-labeled monoclonal antibodies, allowing for simultaneous measurement of [Ca2+], and cell surface staining. Since fluo-3 is a nonratioable calcium indicator, the variability in basal fluorescence intensity compromises the accuracy of flow cytometric measurement of [Ca=+],. However, a significant proportion of variation in fluorescence intensity can be negated by simultaneous analysis of cells loaded with fluo*3 and the intracellular pH indicator SNARF-1. This enables the user to ratio the fluo-3 and SNARF-1 fluorescence intensities, thus yielding a more accurate determination of [Ca2+]l.
Cytometry, 2002
In this study, the effect of antigen-presenting cells (APC), peptide concentration, and CD28 costimulation on calcium signaling, induced by antigen-specific T-cell activation, was studied by flow cytometry. Methods: We used two experimental approaches, which differed in their time scale and in the duration of the T cell-APC interaction, to measure the increase of intracellular free calcium levels ([Ca 2ϩ ] i) in activated T cells: (1) Fluo-3-loaded T cells were activated by cocentrifugation with peptide-loaded APC and the kinetics of fluorescence intensity changes was monitored continuously and (2) peptide-loaded APC and T cells were mixed, cocultured, and the fluorescence intensity was measured at various time intervals. Results: The calcium signal of T cells was dependent on the APC as demonstrated by the ratio of cells exhibiting high versus low fluorescence intensity and by the magnitude of the calcium signal in the activated population. Short-term interaction of T cells with less potent APC or with efficient APC in the presence of low antigen concentration resulted in decreased calcium signaling. CD28mediated costimulation enhanced the magnitude and sustained the increase of intracellular calcium levels. In line with the strong and sustained calcium signals, the activation of the calcium-dependent transcription factors NF-AT, AP-1, and NF-B was induced. Conclusions: Flow cytometric methods, feasible for the rapid and flexible analysis of calcium signaling upon antigen-specific T-cell activation, were established. Kinetics of the increase of mean fluorescence intensity reflected the calcium response of the total cell population whereas statistical analysis of fluorescence intensity at selected time points provided information on the activation state of single cells. Cytometry 47:207-216, 2002.
Journal of Immunological Methods, 2009
In leukocytes, as in many other cell types, cytoplasmic calcium ([Ca 2+ ] i ) changes play a key role in a series of pathways leading to activation. Here we describe a flow cytometric method allowing the simultaneous kinetic analysis of changes in [Ca 2+ ] i in the three types of leukocytes, i.e. monocytes, granulocytes and lymphocytes. Heparinised whole blood was diluted in phosphate buffered saline with Ca 2+ and 1 mM sodium pyruvate and incubated with the Ca 2+ indicator fluo3-acetoxymethyl ester. Leukocytes were identified by labelling with the phycoerythrin-conjugated antibody against CD45, the leukocyte common antigen. Resuspension of the cells in PBS with or without Ca 2+ allowed us to detect the origin of Ca 2+ changes. During flow cytometric analysis only CD45-positive cells were counted and monocytes, granulocytes and lymphocytes were evaluated separately. Baseline fluorescence of the fluo3-Ca 2+ -complex was determined and changes in [Ca 2+ ] i after stimulation with the calcium ionophore A23187 or the chemotactic peptide N-formyl-methionyl-leucylphenylalanine (fMLP) were recorded over a time period of 150 s. Stimulation with A23187 resulted in a rise in [Ca 2+ ] i in all three leukocyte subpopulations. This rise was sustained in the presence of extracellular Ca 2+ (Ca 2+ ex ) but had a transient character in the absence of Ca 2+ ex . For fMLP, [Ca 2+ ] i changes occurred only in monocytes and granulocytes and were transient irrespective of the presence or absence of Ca 2+ ex . In conclusion, the present method is a simple, fast and easy tool to analyse in vitro [Ca 2+ ] i changes over time in leukocytes under physiologically relevant conditions, without the need for their isolation or the lysis of erythrocytes. The whole blood approach allows a continuous interaction between the different leukocyte subpopulations and other blood components and a minimum of preparative manipulations avoids artefactual activation of the cells. A distinction can be made between Ca 2+ release from the intracellular stores and the entry of Ca 2+ from outside the cell. The approach allows to evaluate the effect of various agonists on [Ca 2+ ] i changes in leukocytes, with physiological, patho-physiological or therapeutic purposes.
Methods, 2000
Flow cytometry offers numerous advantages over traditional techniques for measuring intracellular Ca2+ in lymphoid and nonlymphoid cells. In particular, the heterogeneity of cell responses can be defined by flow cytometry, and multiparameter analyses permit the determination of intracellular Ca2+ in surface-marker-defined target cells as well as correlation of changes in Ca2+ with other biochemical markers, including ligand binding. This article presents several established methods for measuring intracellular Ca2+ by flow cytometry in lymphoid and nonlymphoid cells. Examples are provided for determination of Ca2+ in human peripheral blood leukocytes and two human epithelial cell lines grown in monolayer. In addition, applications are reviewed or presented for correlating changes in intracellular Ca2+ with other cell parameters, including cell cycle analysis, changes in cell membrane integrity, and the induction of apoptosis markers. Finally, a number of novel sample handling capabilities useful for performing kinetic analyses of Ca2+ changes by flow cytometry are now available and one application is presented which is finding utility in pharmacologic studies.
Cytosolic calcium determination: a fluorometric technique
The Journal of Nutritional Biochemistry, 1991
The activation of many mammalian cells, including human blood cells, by physiological stimuli evokes a rapid change in cytosolic Ca" ~ levels. The role of calcium as a possible messenger in signal transduction is presently under investigation due to the advent of several new highly fluorescent indicators. The Ca + + fluxes observed in human blood cells-platelets, neutrophils, and monocytes-are rapid, are initiated in milliseconds in some cells, are maximal by 30 seconds, and can be detected with probes such as Quin 2, Fura-2, and Indo-1. Suspension studies using spectrofluorometry, which reflect a value which is the average of all cells, are commonly used: however, recently the adaption of flow cytometry to the measurement of Ca ++ changes in individual cells has been extremely successful. Flow cytometric measurements using a fluorescence activated cell sorter allow analysis of several activation parameters simultaneously, including changes in cytosolic Ca + + and pH, as well as in membrane potential, and also permit correlation of these changes with ligand and antibody binding. Since Ca ........ release into the cytoplasma is one of the earliest signs of cell activation, the adaptation of the fluorescent Indo-I loading technique to other cell types will further the advance of studies seeking to elucidate the role of cytosolic Ca ' + in cell activation. Materials and methods Blood cell isolation Blood was drawn from normal human volunteers and anticoagulated with 0.38% sodium citrate PlateIet isolation Platelets were isolated by gel filtration using a Sepharose 2B column equilibrated with HEPES buffer (137 mM NaCI, 3.3 mM NaH2PO 4, 5.5 mM D-glucose, 2.7 mM KC1, 3.8 mM HEPES, 0.98 mM MgCI 2, and 0.15 Units/ mi apyrase), pH 7,4 as previously described. Leukocyte isolation Leukocytes were isolated from citrated blood by dextran sedimentation followed by Ficoll-Hypaque gradient centrifugation. 2 Polymorphonuclear leukocytes (neutrophils) were separated from contaminating erythrocytes by hypotonic lysis. The resulting cells
Journal of Immunological Methods, 1991
A method is described for flow microfluorometric analysis of calcium mobilization m immune cells. This method is based on dual-color analysis of heterogeneous cell populations using fluo-3 and phycoerythrin (PE)-conjugated monoclonal antibody (mAb). This technique allows the detection of fluo-3 fluorescence as a measure of an increase in cytoplasmic free calcium ([Ca2+]i) while simultaneously discriminating PE-mAb-labeled cells without using wavelength ratio imaging. This method provides a unique way to distinguish patterns of calcium mobilization within a heterogeneous cell population.
Crosstalk between cytosolic pH and intracellular calcium in human lymphocytes
Cellular Signalling, 2000
Stimulation of lymphocytes by specific antigens is followed by the activation of different signal transduction mechanisms, such as alterations in the cytoplasmic levels of Ca 2ϩ , H ϩ and variations in membrane potential. To study interrelationships among these parameters, changes in pHi and Ca 2ϩ were measured with the fluorescent probes BCECF and Fura-2 in freshly isolated blood human lymphocytes. Moreover, membrane potential qualitative alterations were recorded with the fluorescent dye bisoxonol. In a bicarbonate-free medium, cell alkalinization with NH 4 Cl slightly decreased intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i) due to efflux of Ca 2ϩ from the cell. In contrast, an elevation of pHi induced with 4-AP increased [Ca 2ϩ ] i , either in the presence or absence of external Ca 2ϩ. The increase in Ca 2ϩ-free medium is likely to be due to Ca 2ϩ release from thapsigargin and caffeineindependent intracellular stores. Both 4-AP or NH 4 Cl induced a plasma membrane depolarisation, although with different kinetics. The ionosphere ionomycin increased pHi, Ca 2ϩ levels and also induced membrane depolarisation. Together, these observations demonstrate a lack of correlation between the magnitude of changes in pHi and Ca 2ϩ .
Calcium response of helper T lymphocytes to antigen-presenting cells in a single-cell assay
Biophysical Journal, 1995
We developed a dynamic, single-cell assay involving alternating differential interference contrast and fluorescence microscopy, together with digital imaging, for both viewing the physical interaction of live helper T lymphocytes (Th cells) with antigen-presenting cells (APCs) and monitoring the increases in the intracellular free calcium concentration of the Th cell, an early event in Th cell activation. We obtained Th-APC conjugates by allowing the Th cells to migrate toward and interact with APCs that either settled nearby or had been micromanipulated in close proximity to the Th cells. Th cell motility played an important role in initiating Th-APC contacts but not in determining the Th cell calcium response. We found that the intracellular calcium responses of individual Th cells are heterogeneous and an all-or-none phenomenon, independent of antigen concentration. However, the fraction of Th-APC conjugates involving responding Th cells is an increasing function of the antigen concentration. Finally, we measured some characteristics of the developing Th-APC contact area. We used all of these data together with previously developed mathematical models to estimate that only 1 to 20 major histocompatibility class Il-antigen complexes are required in the initial Th-APC contact area to elicit a Th cell calcium response.
Calcium-dependent activation of T-lymphocytes
Pfl�gers Archiv - European Journal of Physiology, 2005
Activation of T-lymphocytes requires stimulation of T-cell receptors (TCR) and co-stimulatory signals. Among different signalling cascades, TCR engagement induces Ca 2+ entry through plasma membrane Ca 2+ channels, which is an indispensable step for T-cells to expand clonally and to acquire effector functions. The Ca 2+ channels are activated by depletion of Ca 2+ stores and are called Ca 2+ release-activated Ca 2+ (CRAC) channels. Ca 2+ influx through CRAC channels is also controlled, directly or indirectly, by K + channels, Ca 2+ -ATPases, mitochondria, endoplasmic reticulum and Ca 2+ buffers. We review the functional implications of these transporters, organelles and buffers and develop a model of Ca 2+ signal generation that depends mainly on their relative mutual localization. This model offers the possibility of controlling amplitude and kinetics of Ca 2+ signals in T-cells. Decoding of various Ca 2+ signals allows differential activation of the transcription factor families nuclear factor of activated T-cells (NFAT), nuclear factor-jB (NF-jB) and activator protein-1 (AP-1). Variation of amplitude and kinetics of Ca 2+ signals thus is an important mechanism for modulating the specificity of T-cell responses.