Probing cell proliferation: Considerations for dye selection.

Broadly speaking, cell tracking dyes are fluorescent compounds that bind stably to components on or within the cells so the fate of the labeled cells can be followed. Their staining should be bright and homogeneous without affecting cell function. For purposes of monitoring cell proliferation, each time a cell divides the intensity of cell tracking dye should diminish equally between daughter cells. These dyes can be grouped into two different classes. Protein reactive dyes label cells by reacting covalently but non-selectively with intracellular proteins. Carboxyfluorescein diacetate succinimidyl ester (CFSE) is the prototypic general protein label. Membrane intercalating dyes label cells by partitioning non-selectively and non-covalently within the plasma membrane. The PKH membrane dyes are examples of lipophilic compounds whose chemistry allows for their retention within biological membranes without affecting cellular growth, viability, or proliferation when used properly. Here we provide considerations based for labeling cell lines and peripheral blood mononuclear cells using both classes of dyes. Examples from optimization experiments are presented along with critical aspects of the staining procedures to help mitigate common risks. Of note, we present data where a logarithmically growing cell line is labeled with both a protein dye and a membrane tracking dye to compare dye loss rates over 6days. We found that dual stained cells paralleled dye loss of the corresponding single stained cells. The decrease in fluorescence intensity by protein reactive dyes, however, was more rapid than that with the membrane reactive dyes, indicating the presence of additional division-independent dye loss.

Monitoring Cell Proliferation by Dye Dilution: Considerations for Panel Design.

High dimensional studies that include proliferation dyes face two inherent challenges in panel design. First, the more rounds of cell division to be monitored based on dye dilution, the greater the starting intensity of the labeled parent cells must be in order to distinguish highly divided daughter cells from background autofluorescence. Second, the greater their starting intensity, the more difficult it becomes to avoid spillover of proliferation dye signal into adjacent spectral channels, with resulting limitations on the use of other fluorochromes and ability to resolve dim signals of interest. In the third and fourth editions of this series, we described the similarities and differences between protein-reactive and membrane-intercalating dyes used for general cell tracking, provided detailed protocols for optimized labeling with each dye type, and summarized characteristics to be tested by the supplier and/or user when validating either dye type for use as a proliferation dye. In this fifth edition, we review: (a) Fundamental assumptions and critical controls for dye dilution proliferation assays; (b) Methods to evaluate the effect of labeling on cell growth rate and test the fidelity with which dye dilution reports cell division; and. (c) Factors that determine how many daughter generations can be accurately included in proliferation modeling. We also provide an expanded section on spectral characterization, using data collected for three protein-reactive dyes (CellTrace™ Violet, CellTrace™ CFSE, and CellTrace™ Far Red) and three membrane-intercalating dyes (PKH67, PKH26, and CellVue® Claret) on three different cytometers to illustrate typical decisions and trade-offs required during multicolor panel design. Lastly, we include methods and controls for assessing regulatory T cell potency, a functional assay that incorporates the "know your dye" and "know your cytometer" principles described herein.

Monitoring Cell Proliferation by Dye Dilution: Considerations for Probe Selection.

In the third edition of this series, we described protocols for labeling cell populations with tracking dyes, and addressed issues to be considered when combining two different tracking dyes with other phenotypic and viability probes for the assessment of cytotoxic effector activity and regulatory T cell functions. We summarized key characteristics of and differences between general protein and membrane labeling dyes, discussed determination of optimal staining concentrations, and provided detailed labeling protocols for both dye types. Examples of the advantages of two-color cell tracking were provided in the form of protocols for: (a) independent enumeration of viable effector and target cells in a direct cytotoxicity assay; and (b) an in vitro suppression assay for simultaneous proliferation monitoring of effector and regulatory T cells.The number of commercially available fluorescent cell tracking dyes has expanded significantly since the last edition, with new suppliers and/or new spectral properties being added at least annually. In this fourth edition, we describe evaluations to be performed by the supplier and/or user when characterizing a new cell tracking dye and by the user when selecting one for use in multicolor proliferation monitoring. These include methods for: (a) Assessment of the dye's spectral profile on the laboratory's flow cytometer(s) to optimize compatibility with other employed fluorochromes and minimize compensation problems; (b) Evaluating the effect of labeling on cell growth rate;

Optimized staining and proliferation modeling methods for cell division monitoring using cell tracking dyes.

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.(1-5) Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of: stem and progenitor cell quiescence, proliferation and/or differentiation(6-8) antigen-driven membrane transfer(9) and/or precursor cell proliferation(3,4,10-18) and immune regulatory and effector cell function(1,18-21). Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes(1,2,11). "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins(4,16,18). Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class(2-4,16,18,24). The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are: Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies. Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 10(2) - 10(3) times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used. Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile. Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Tracking immune cell proliferation and cytotoxic potential using flow cytometry.

In the second edition of this series, we described the use of cell tracking dyes in combination with tetramer reagents and traditional phenotyping protocols to monitor levels of proliferation and cytokine production in antigen-specific CD8(+) T cells. In particular, we illustrated how tracking dye fluorescence profiles could be used to ascertain the precursor frequencies of different subsets in the T-cell pool that are able to bind tetramer, synthesize cytokines, undergo antigen-driven proliferation, and/or carry out various combinations of these functional responses.Analysis of antigen-specific proliferative responses represents just one of many functions that can be monitored using cell tracking dyes and flow cytometry. In this third edition, we address issues to be considered when combining two different tracking dyes with other phenotypic and viability probes for the assessment of cytotoxic effector activity and regulatory T-cell functions. We summarize key characteristics of and differences between general protein- and membrane-labeling dyes, discuss determination of optimal staining concentrations, and provide detailed labeling protocols for both dye types. Examples of the advantages of two-color cell tracking are provided in the form of protocols for (a) independent enumeration of viable effector and target cells in a direct cytotoxicity assay and (b) simultaneous monitoring of proliferative responses in effector and regulatory T cells.

Tracking antigen-driven responses by flow cytometry: monitoring proliferation by dye dilution.

Cell-tracking reagents such as the green-fluorescent protein labeling dye CFSE and the red-fluorescent lipophilic membrane dye PKH26 are commonly used to monitor cell proliferation by flow cytometry in heterogeneous cell populations responding to immune stimuli. Both reagents stain cells with a bright homogeneous fluorescence, which is partitioned between daughter cells during each cell division. Because daughter cell fluorescence intensities are approximately halved after each division, the intensity of a cell relative to its intensity at the time of staining provides information about how many divisions it has undergone. Knowing how many rounds of division have occurred and the relative number of cells in each daughter generation, one can back-calculate the number of cells in the original population (i.e., cells present at the time of stimulus) that went on to respond by proliferating. Using this information, the precursor cell frequencies and extent of expansion to a specific antigen or mitogen of interest can be calculated. Concurrently, the phenotype of the cells can be determined, as well as their ability to bind antigen or synthesize cytokines, providing more detailed characterization of all cells responding to the antigen, not just effector cells. In multiparameter flow cytometric experiments to simultaneously analyze antigen-specific tetramer binding, cytokine production and T-cell proliferation, we found that only approximately half of the cells that exhibited specific binding to influenza tetramer also proliferated, as measured by dye dilution, and synthesized IFNgamma in response to antigen. We expect the advent of new cell tracking dyes emitting from the violet to the near infrared combined with the increasing number of lasers and detectors on contemporary flow cytometers to further expand the usefulness of this approach to characterization of complex antigen-driven immunological responses.

Cell tracking 2007: a proliferation of probes and applications.

The articles in this thematic issue, entitled "Tracking Cell Proliferation and Function," illustrate some of the choices made by authors pushing the envelope for cell tracking applications in their areas of interest. Over the past decade there has been a proliferation in the range of commercially available probes for these studies, the capabilities of the instrumentation used to detect them, and in the biological systems being studied. This introductory to the thematic issue presents the advantages and limitations of the more commonly used probes such as CFSE and PKH26, as well as emerging probes that expand the range of fluorescence available, including quantum dots and the new CellVue dyes. Appropriate method and instrument setup controls and possible data analysis strategies are discussed with the goal of urging experienced investigators to include all critical information and controls when publishing their data and of aiding researchers new to cell tracking to make informed decisions on which cell tracking reagent(s) are best suited for their particular application. All cell tracking assays have the common goal of determining the fate of a particular cell population within a heterogeneous environment, whether in vivo or in vitro. Some of the common themes among the contributions found in this issue include how various probes are used to track (i) cell proliferation, (ii) regulatory and effector immune cell function and (iii) membrane transfer and antigen presentation. Although these represent only a small fraction of the large and growing list of applications for cell tracking, clearly illustrate the growing trend toward the use of multiple tracking reagents and multiple detection modalities to address complex biological questions.

CellVue Claret, a new far-red dye, facilitates polychromatic assessment of immune cell proliferation.

Flow cytometric analyses of immune cell proliferation, differentiation, and function are limited by the number of different fluorochromes that can be resolved simultaneously. Additional colors to expand functional analytic capability will facilitate higher dimensional analyses of heterogeneous cell populations by basic and clinical scientists. Our aim in these studies was to evaluate CellVue Claret, a fluorescent, far-red emitting, membrane intercalating dye (excitation maximum: 655 nm, emission maximum 677 nm), as an alternative and/or complementary probe to PKH26 and CFSE(1) for polychromatic studies of immune cell proliferation and function. Using a BD FACSCalibur and human peripheral blood mononuclear cells (PBMCs) from 8 different donors (2 donors studied twice), we compared CellVue Claret with the two most commonly used visible-emitting proliferation dyes, PKH26 and CFSE, in terms of: (1) compatibility with 7-Amino-actinomycin D (7-AAD) as a viability marker; (2) effect of dye labeling on lymphocyte viability; and (3) the proliferative response of CD3+ T lymphocytes from 0-96 hours as assessed by dilution of each of the 3 cell tracking dyes in cultures stimulated with anti-CD3 plus IL-2. Post-labeling recoveries and viabilities were similar for all 3 dyes, with modestly higher initial staining intensities and coefficients of variation for CellVue Claret than for CFSE or PKH26. Lymphocyte viabilities in stimulated or unstimulated cultures were also unaffected by choice of dye. Proliferative responses of viable CD3+ lymphocytes were comparable for all three dyes, whether results were reported as Proliferative Fraction (percent of cells that had divided one or more times) or as Precursor Frequency (percent of parent population that had gone on to proliferate in response to anti-CD3 plus IL-2). In summary, T cell proliferation analysis using CellVue Claret gives results equivalent to those obtained with PKH26 or CFSE, expanding the choice of proliferation dyes suitable for use in high dimensional polychromatic studies on flow cytometers with far red (633 nm-658 nm) excitation capabilities.

Novel lipophilic tracking dyes for monitoring cell proliferation.

The advent of contemporary digital instrumentation has enhanced both the potential and the complexity of flow cytometric experiments, allowing for the detailed dissection of immune cell subsets and their functions. The use of cell tracking labels such as PKH26 and CFSE has been important in observing such cellular functions, but their visible emission characteristics have limited the design of such analyses. As the demand for multiparametric flow cytometry intensifies, it will become increasingly important to utilize a broader range of cell tracking reagents to optimize the measurement of fluorescence signals and to provide flexibility in the use of commercially available fluorochrome - antibody combinations. We report on the evaluation of three lipophilic membrane dyes, CellVue Lavender, CellVue Plum and CellVue NIR780; with fluorescence emissions in the violet, far-red and near infrared wavelength regions, respectively. These reagents are similar to established tracking dyes such as PKH26 and CFSE in terms of staining procedure, membrane stability, optimal concentration, and lack of effect on cellular proliferation. The CellVue dyes however, exhibit different spectral characteristics than existing tracking compounds, and capitalize upon the increased number of lasers incorporated into commercially available instrumentation; thus permitting measurement of labeled populations in underexploited regions of the spectrum.