While regulated cell proliferation provides the foundation for normal growth and development, cell cycle dysregulation is a hallmark of cancer and autoimmune diseases. The use of high-throughput, fluorescence-based methods such as flow cytometry is paramount to the study of cell proliferation. Key to proliferation analysis is the ability to uniformly label live cells with a fluorescent dye and then clearly identify distinct generations of cells through time by following the decrease in fluorescent signal as it is diluted in daughter cells. Without adequate resolution of the division peaks (Figure 1), it is difficult to assess quantitative cell proliferation data accurately using dye dilution, even with the use of fitting algorithms.

The cell tracing dye carboxyfluorescein diacetate succinimidyl ester (CFDA SE, or CFSE) is among the earliest and most popular reagents used in cell cycle studies [1]. With the development of CFSE alternatives that do not require 488 nm excitation, cell proliferation assays are now more amenable to multiplex analyses with other fluorescent probes. Filby and coworkers recently published a method to assess and compare the performance of five commercially available fluorescent cell tracing reagents: three succinimidyl ester–based dyes (Invitrogen™ CellTrace™ Violet and CellTrace™ Far Red dyes and Affymetrix™ Cell Proliferation Dye eFluor™ 670) and two lipophilic dyes (CellVue™ Claret and PKH26 reagents), none of which requires 488 nm excitation. Jurkat cells were used in this study because these transformed heterogeneous, nonquiescent cells provide a more challenging model system than relatively uniform primary T and B cells and, when labeled, invariably exhibit a broad fluorescence distribution. For each of the dyes, performance testing included:

  • Labeling efficiency, via measurement of viability, intensity, and uniformity of the dye-labeled cells
  • Need for cell sorting to resolve division peaks
  • Dye-specific contribut ion to any re-spreading error of the sorted populations
  • Nonspecific cell-to-cell dye transfer
  • Symmetrical inheritance across the cytokinetic plane

The authors observed that, although culture-dependent sources of error exist, succinimidyl ester–based dyes were superior for cell proliferation studies when compared with lipophilic dyes. Lipophilic dyes required higher labeling concentrations to achieve comparable staining intensity and uniformity, and they produced more culture-dependent variation that impacted peak resolution. However, except for CellTrace Violet dye, the succinimidyl ester–based dyes also demonstrated various suboptimal attributes. CellTrace Violet is the only dye that did not transfer between cells in culture; moreover, CellTrace Violet distributed across the cytokinetic plane in a highly symmetrical fashion, resulting in well-resolved division peaks. The authors state, “Collectively, these data support our current view that of the dyes we have tested, [CellTrace Violet dye] still remains the best for tracking proliferation in any amenable cell type by fluorescent dye dilution and flow cytometry.” This conclusion is in line with an earlier study by Quah and coworkers [1] that also found CellTrace Violet dye to be “the best available alternative to CFSE in the analysis of cell divisions.”

In conclusion, Filby and colleagues suggest that the choice of labeling dye and, when necessary, the use of cell sorting to narrow the width of the parent cell population are critical steps when conducting cell proliferation experiments. These steps should ensure that division peaks can be well resolved and that any algorithms applied to the data will generate accurate cell parameters, including percentage of divided cells and proliferation index.

Figure 1. Tracking cell division in lymphocytes using CellTrace Violet dye. Human peripheral blood lymphocytes were harvested and stained with CellTrace™ Violet dye. The violet peaks represent successive generations of cells stimulated with anti–human CD3 antibody and interleukin-2 (IL-2) and cultured for 7 days. The peak outlined in black represents cells that were cultured for 7 days with no stimulus. Data were collected on a BD™ LSR II Cell Analyzer and analyzed using Attune™ cytometer software. Reproduced from The Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies, 11th edition (2010).

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