Using the Attune® Cytometer and CellTrace™ Violet Dye
Successful cell proliferation analysis by dye dilution requires sensitive instrumentation and an extremely bright dye to accurately distinguish fluorescently labeled cells from autofluorescence after several cell divisions. The combination of the Attune® Acoustic Focusing Cytometer and Molecular Probes® CellTrace™ Violet dye allows the identification of up to 10 population doublings following stimulation. And, because CellTrace™ Violet dye emission is collected from the violet laser of the Attune® cytometer, it is fully compatible with Green Fluorescent Protein (GFP)–expressing cells for additional multiplexing capabilities.
Tracing Cell Division Using the Attune® Cytometer
Ten cell divisions were identified with the Attune® Acoustic Focusing Cytometer and the Molecular Probes® CellTrace™ Violet Cell Proliferation Assay using human peripheral blood mononuclear cells. Figures below demonstrate that the Attune® cytometer resolves population peaks that are close together (Figure 1A and 1B) and simplifies multiplexing with GFP cells (Figure 1C).
Figure 6. Ten cell divisions identified with the Attune® Acoustic Focusing Cytometer and the Molecular Probes® CellTrace™ Violet Cell Proliferation Assay. Human peripheral blood mononuclear cells were isolated from whole blood, stained with CellTrace™ Violet, and stimulated to proliferate in culture. Cells were stained with mouse anti–human CD4 Alexa Fluor® 488 prior to analysis on the Attune® Acoustic Focusing Cytometer at a flow rate of 25 μL/min. (A) Histogram of fluorescence intensity with each peak representing one subsequent generation of proliferating cells. (B) To provide statistics about each generation of cells in a population, the fluorescence histogram was further analyzed with proliferation modeling software (ModFit LT™ software, Verity Software House). The location of each generation of cells is represented by a unique peak color. (C) Two-dimensional plot allowing the simultaneous analysis of cell proliferation between CD4+ and CD4- cell populations.