Search Thermo Fisher Scientific
- Order Status
- Quick Order
-
Don't have an account ? Create Account
Search Thermo Fisher Scientific
Live cell studies of cellular DNA content and cell cycle distribution are useful to detect variations of growth patterns due to a variety of physical, chemical, or biological means, to monitor apoptosis, and to study tumor behavior and suppressor gene mechanisms. In a given population, cells are distributed among three major phases of cell cycle: G0/G1 phase (one set of paired chromosomes per cell), S phase (DNA synthesis with variable amount of DNA), and G2/M phase (two sets of paired chromosomes per cell, prior to cell division).1–4 DNA content can be measured using fluorescent, DNA-selective stains that exhibit emission signals proportional to DNA mass. Flow cytometric analysis of these stained populations is then used to produce a frequency histogram that reveals the various phases of the cell cycle. This analysis is typically performed on permeabilized or fixed cells using a cell-impermeant nucleic acid stain, but is also possible using live cells and a cell-permeant nucleic acid stain. While the choices for fixed cell staining are varied, there are only a few examples of useful cell-permeant nucleic acid stains.
The Vybrant™ DyeCycle™ Violet stain is a DNA-selective, cell membrane-permeant, and nonfluorescent stain that uses the violet laser for DNA content analysis in living cells. The Vybrant DyeCycle Violet stain is fluorescent upon binding to double-stranded DNA. Well suited for the popular violet laser line (Figure 1), Vybrant DyeCycle Violet stain can also be used with UV excitation, having emission at ~440 nm.
The staining protocol is simple and includes incubating suspended cells in the presence of Vybrant DyeCycle Violet stain and directly measuring the fluorescence without the need for any additional treatment or centrifugation steps. This live cell stain allows the simultaneous co-staining of the cell population for other parameters, and allows for the possibility of cell sorting based on DNA content. Vybrant DyeCycle Violet stain does efflux in rodent and human stem cells, making the Side Population (SP) technique now available with violet excitation.5
Spectral Characteristics
The fluorescence excitation and emission spectra of the stain are shown in Figure 1. The spectra were obtained from samples of the Vybrant DyeCycle Violet stain bound to DNA. The Vybrant DyeCycle Violet stain /DNA complex has fluorescence excitation and emission maxima of 369/437 nm, respectively.
![]() | Figure 1. Fluorescence excitation and emission spectra for the Vybrant DyeCycle Violet stain bound to DNA in TBE,pH 8.3. |
Table 1. Contents and storage information.
Material | Amount | Concentration | Storage* | Stability | |
---|---|---|---|---|---|
Vybrant DyeCycle Violet stain | 200 μL | 5 mM solution in deionized water |
| When stored as directed, this kit is stable for at least 6 months. |
Number of assays: Sufficient material is supplied for approximately 200 flow cytometry assays based on a 1 mL test volume.
Approximate fluorescence excitation/emission maxima:Vybrant DyeCycle Violet stain: 369/437 in nm bound to DNA.
Materials Required but Not Provided
Caution
The hazards posed by this stain have not been fully investigated. Since Vybrant DyeCycle Violet stain is known to bind to nucleic acids, treat the stain as a potential mutagen and use with appropriate care. The stain is supplied as a solution in DMSO, which is known to facilitate the entry of organic molecules into tissues. Use the stain using equipment and practices appropriate for the hazards posed by such materials. Dispose of the reagents in compliance with all pertaining local regulations.
The following staining protocol was optimized using Jurkat cells, a human T-cell leukemia line, in complete RPMI medium containing 10% fetal bovine serum with staining at 37˚C, but can be adapted to most cell types. Test samples comprise of 1 × 106 cells per 1 mL. Growth medium or buffer used, cell density, cell type variations, and other factors may influence staining. In initial experiments, try a range of dye concentrations to determine the one that yields optimal staining for the given cell type, buffer, and experimental condition. For a given experiment, each flow cytometry sample should contain the same number of cells, as sample-to-sample variation in cell number leads to significant differences in fluorescence signal.
If Vybrant DyeCycle Violet stain is used in combination with other stains for multicolor applications, apply the other stain(s) to the sample first, following all manufacturers’ instructions, including wash steps. Vybrant DyeCycle Violet stain should be the last stain applied to the sample, and do not wash or fix samples prior to flow cytometric analysis.
General Guidelines
For optimal DNA content cell cycle analysis, follow these guidelines:
Vybrant DyeCycle Violet Staining Protocol
This basic protocol is optimized using Jurkat cells suspended in complete medium (RPMI/10% fetal bovine serum) and stained with Vybrant DyeCycle Violet stain at 37˚C.
![]() | Figure 2. Histogram of live Jurkat cells stained with Vybrant DyeCycle Violet stain showing DNA content distribution. G0/G1 and G2/M phase histogram peaks are separated by the S-phase distribution. Violet 405 nm excitation was used with a 440/40 nm bandpass filter. |