Dyes of the other class, here referred to as “membrane dyes”, stably intercalate into the lipid bilayer of cell membranes via strong hydrophobic associations. Dyes of one class, here referred to as “protein dyes”, react with proteins to form a covalent bond. Although diverse in their chemistries and fluorescence characteristics, these reagents can be grouped into two main classes based upon their mechanism of cell labeling. The number of fluorescent dyes commercially available for cell tracking, and the subset useful for proliferation monitoring (reviewed in ), continues to expand rapidly, with new suppliers and/or new spectral properties continually being added.

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. 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. 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. 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. 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.

Flowjo x proliferation