Much of our current knowledge of the immune system derives from the ability to initiate an ex vivo immune response in isolated T lymphocytes, triggering their proliferation. A common technique for quantifying this proliferation is using a fluorescent label that is divided evenly between two daughter cells following cell division (Figure 1). CellTrace™ Violet is a novel cell proliferation tracing dye with a mode of action similar to carboxyfluorescein diacetate succinimidyl ester (CFSE).
CellTrace™ Violet easily diffuses into cells, where it is cleaved by intracellular esterases to yield a highly fluorescent compound. This compound covalently binds to intracellular amines, resulting in stable, well-retained fluorescent staining that can be fixed with aldehyde fixatives. Excess unconjugated reagent diffuses to the extracellular medium, where it can be quenched with complete medium and washed away.
Figure 1. Bright, well-retained dye that partitions evenly between daughter cells facilitates proliferation analysis. (Left) Illustration of proliferation analysis by dye dilution. (Right) Flow cytometric analysis reveals a bright, homogeneous fluorescent signal from the initial population of cells. Subsequent cell divisions result in larger numbers of cells, each with half the fluorescence intensity of its parent cell.
Superior Fluorescent Staining
Successful proliferation analysis by dye dilution requires an extremely bright dye to distinguish fluorescently labeled cells from autofluorescence after several cell divisions. The intense fluorescent staining provided by CellTrace™ Violet enables the visualization of ten or more generations of proliferating cells before the signal is overwhelmed by intrinsic cellular autofluorescence. Consistent, homogeneous staining results in very little fluorescence variation between cells in a population, so distinct generations can be seen without any requirement for complex modeling software.
Long-Term Signal Stability
Some fluorescent dyes for proliferation analysis quickly lose a large percentage of their fluorescent signal after staining, requiring the researcher to wait for the signal to stabilize before stimulating cells. CellTrace™ Violet shows only a small drop in initial fluorescence. CellTrace™ Violet easily crosses the plasma membrane and covalently binds inside cells where the stable, well-retained fluorescent dye provides a consistent signal, even after several days in a cell culture environment. CellTrace™ Violet also shows little cytotoxicity, with minimal observed effect on the proliferative ability or biology of cells.
Simple, Robust Staining Protocol
The CellTrace™ Violet Cell Proliferation Kit contains convenient single-use vials of dry dye to permit small-scale experiments without preparing excess quantities of dye. A stock solution is prepared by dissolving the contents of a vial in anhydrous DMSO prior to use. To stain 1 mL of cells in protein-free medium, 1 µL of this stock solution is typically used. Cells should be stained for 20 minutes at room temperature with gentle agitation. A brief wash with complete medium will then quench any dye remaining in solution.
Easy Multiplexing With Other Fluorophores
Modern instrumentation permits the analysis of many fluorescent parameters in a single flow cytometry experiment. The violet excitation and narrow emission of CellTrace™ Violet permit multiplexing with most blue-, green-, or red-excitable fluorophores, including Alexa Fluor® 488, FITC, and R-phycoerythrin, as well as in cells expressing Green Fluorescent Protein (GFP).
CellTrace™ Violet can be used in multicolor flow cytometry experiments to precisely track the proliferation of cells in culture while collecting other data. To demonstrate this application, the superior resolution of the new Attune™ Acoustic Focusing Cytometer was used in a multicolor experiment with CellTrace™ Violet to track ten generations of proliferating lymphocytes (Figure 2). For more information on the Attune™ cytometer, read the article on The Attune™ Acoustic Focusing Cytometer.
Learn more about the CellTrace™ Violet Cell Proliferation Kit at CellTrace™ Reagents for Cell Proliferation.
Figure 2. Multiparameter proliferation analysis of human lymphocytes. Peripheral blood mononuclear cells were isolated from whole blood, stained with 10 µM CellTrace™ Violet, and stimulated with mouse anti–human CD3 and interleukin-2 for 7 days in culture. Cells were stained with SYTOX® AADvanced™ Dead Cell Stain and mouse anti–human CD4 Alexa Fluor® 488 immediately prior to analysis on the Attune™ Acoustic Focusing Cytometer. A gating strategy was employed to limit analysis to live cells (A), single cells (B), and CD4+ cells (C). When the resulting population of cells is displayed on a histogram of CellTrace™ Violet fluorescence intensity (D), each peak represents one generation of proliferating cells. This fluorescence histogram can be further analyzed using proliferation modeling software (ModFit LT™, Verity Software House) to provide statistics and uniquely identify each generation of cells with a different peak color (E).
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