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;
Keywords: Cell division; Cell tracking; CellTrace™ dyes; CellVue® dyes; Cytotoxicity; Dye dilution proliferation assay; Flow cytometry; PKH dyes.
(c) Testing the fidelity with which dye dilution reports cell division; (d) Determining the maximum number of generations to be included when using dye dilution profiles to estimate fold population expansion or frequency of responder cells; and (e) Verifying that relevant cell functions (e.g., effector activity) remain unaltered by tracking dye labeling.