The Vybrant® DyeCycle™ family of dyes offers the ability to stain for DNA profiling in live cells with options for using 405, 488, 532, or 633 nm excitation. The dyes have low cytotoxicity, allowing stained cells to be sorted and otherwise cultured or assessed with functional assays after staining.
Vybrant® DyeCycle™ Stains Selection Guide
How Vybrant® DyeCycle™ Stains Work
Vybrant® DyeCycle™ stains for flow cytometry are ideal tools for DNA content analysis in living cells. These DNA-selective stains are cell membrane permeant, and after binding DNA emit a fluorescence signal that is proportional to DNA mass.
Fluorescence data can be used to generate a histogram showing the distribution of cells in the phases of the cell cycle. These live-cell dyes allow the simultaneous co-staining of the cell population for other parameters, and due to their low cytotoxicity provide the possibility of cell sorting based on DNA content.
|Figure 1. Cell cycle analysis using Vybrant® DyeCycle™ Violet stain. Histogram of live Jurkat cells stained with Vybrant® DyeCycle™ Violet stain showing DNA content distribution. G0/G1 and G2/M peaks are separated by the S phase distribution. Violet 405 nm excitation was used with a 440/40 nm bandpass filter.|
Why use Vybrant® DyeCycle™ stains?
Ability to combine cell cycle analysis with other live cell applications
Combining cell cycle analysis with additional live cell applications (i.e., analysis of GFP cells, immunophenotyping, cell sorting, CFSE cell tracing, and apoptotic sub-G1 population analysis) is a snap with Vybrant® DyeCycle™ stains. These stains are cell-permeant nucleic acid stains that can penetrate the nucleus without cell fixation therefore, they are compatible with many live cell applications.
Multiple color options for easier multiplexing
Vybrant® DyeCycle™ stains are available in four different color choices for use on UV, violet, blue, green, or red lasers. These options allow researchers to free up other channels on their flow cytometers for additional parameters in their experiments. DyeCycle™ Ruby takes advantage of the commonly available 488 nm and 633/5 nm excitation sources with emission >670 nm, placing cell cycle studies within reach of all flow cytometrists.
Ability to sort based on phase of cell cycle
Vybrant® DyeCycle™ stains exhibit relatively low cytotoxicity, allowing the possibility of sorting based on phase of the cell cycle. Recently, Vybrant® DyeCycle™ Violet was used to sort adult subventricular zone neural stem cell populations according to their stage in the cell cycle (Pastrana, et al. PNAS; (106) 6387-6392.)
Ability to identify stem cell side populations on the violet laser
Vybrant® DyeCycle™ Violet Stain has also been shown to identify stem cell side populations and early progenitors in mammalian hematopoietic tissues using the violet laser. Isolated cells were shown to have the same phenotypic characteristics of SP cells isolated with Hoechst 33342 (Telford, et al. (2006) Stem Cells 25:1029–1036).
|Figure 2. Analysis of stem cell side population in human cord blood using Dycle™ Violet stain. Cells were incubated with 10 µM DyeCycle™ Violet stain for 90 min at 37°C, then washed and stored on ice until analysis. Violet laser excitation showed a recognizable side population of cells (red bracket). Data courtesy of Dr. William Telford, NIH.|
Vybrant® DyeCycle™ Ruby stain shows significantly less cytotoxicity than DRAQ5, with cytotoxicity levels similar to commonly used Hoechst 33342.
Figure 3. Brightfield images of cells grown after sorting. Live Jurkat cells were stained with either 5 µM UV-excitable Hoechst 33342, 5 µM Vybrant® DyeCycle™ Ruby stain, or 5 µM DRAQ5 DNA dye. The cells were then sorted and cultured for 8 days to examine their ability to grow after staining and sorting. Visual inspection of the cultures show the characteristic grape-like clusters of growing Jurkat cells, in the cells stained with Hoechst 33342 and Vybrant® DyeCycle™ Ruby stains, comparable to the control cells. The cells stained with DRAQ5 dye did not show growth, clearly demonstrating the DRAQ5 dye is cytotoxic.