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.

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.

Flow cytometry analysis emerges as an alternative methodology to microscopy for determination of the Leishmania-infection rates of macrophages. Various flow cytometric approaches have been established for the quantification of Leishmania parasites within host cells, labelled either directly fluorescent dyes or indirectly with fluorescently conjugated antibodies. Although these techniques allow accurate quantification of infection, they fail at detection of non-infected macrophages specifically. This study introduces a new flow cytometric approach for the determination of infection rates of macrophages infected by Leishmania infantum parasites. Prior to infection, J774A.1 macrophages and L. infantum promastigotes were stained separately with PKH26 and PKH67 dyes, respectively. Dual staining enabled detection of each cell type, where non-infected macrophages were also recorded for the quantification. Dual-PKH staining achieved high success in selective staining of promastigotes (99.71%) and macrophages (99.57%). The percentages of parasite-infected macrophages were determined for initial 1:2.5 and 1:10 infection ratios as 15.68 and 61.70%, respectively; indicating significant increase in infection rate parallel to the initial treatment ratio. These results demonstrated that the introduced dual-fluorescence flow cytometric approach can be successfully used as an accurate and rapid quantification method for L. infantum-infected macrophages and strengthens the hypothesis that flow cytometric approaches could replace conventional microscopic methodologies.

Extracellular vesicles (EVs) are mediators of intercellular communication in diverse cellular functions. Visualizing EVs in vivo is important to elucidate the biogenesis of EVs, and various approaches have been developed for in vivo EV tracking. The ubiquitously expressed tetraspanin CD63 is liberally incorporated into EVs. Thus, fluorescently tagged CD63 has been used in many studies to label EVs. In the present study, we presented isolation and transfer assays for EVs from two transgenic rats expressing CD63-GFP in their body fluids or brains.

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;