CellTrace™ Violet Cell Proliferation Kit is used for in vitro and in vivo labeling of cells to trace multiple generations using dye dilution by flow cytometry.• Superior performance—bright, single-peak staining enables visualization of multiple generations• Long-term signal stability—well-retained in cells for several days post stain• Versatile—multiple colors available to easily combine with antibodies or markers of cell function, such as GFP• Simple, robust staining protocolSuperior fluorescent stainingSuccessful proliferation analysis by dye dilution (see figure below) requires an extremely bright dye to distinguish fluorescently labeled cells from auto-fluorescence after several cell divisions. The intense fluorescent staining provided by CellTrace™ Violet dye enables the visualization of ten or more generations of proliferating cells before the signal is overwhelmed by intrinsic cellular auto-fluorescence. 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 retentionUnlike stains that label the lipid membrane of cells, CellTrace™ Violet stain 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 dye binds covalently to all free amines on the surface and inside of cells and shows little cytotoxicity, with minimal observed effect on the proliferative ability or biology of cells.Easy multiplexing with other fluorophoresThe violet excitation and narrow emission of CellTrace™ Violet dye make it ideal for multiplexing due to the limited spectral overlap with other common dyes (Alexa Fluor™ 488, FITC, and RPE) and fluorescent proteins (Green Fluorescent Protein (GFP) and mCherry) (see Fluorescence Spectra for CellTrace™ Violet stain below).Simple, robust staining protocolThe 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.