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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 st 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 cell cycle phases. 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™ Green and Orange stains are DNA-selective, cell membrane-permeant, and nonfluorescent stains for DNA content analysis in living cells. The Vybrant DyeCycle Green and Orange stains are fluorescent upon binding to double-stranded DNA. These stains take advantage of the commonly available 488 nm excitation source, placing cell cycle studies on live cells within reach of all flow cytometrists. Vybrant DyeCycle Green stain is excited at 488 nm with emission ~520 nm (Figure 1). Vybrant DyeCycle Orange stain is excited using both 488 nm and 532 nm laser lines with emission ~570 nm (Figure 2).
The staining protocol is simple and includes incubating suspended cells in the presence of Vybrant DyeCycle 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.
Spectral Characteristics
The fluorescence excitation and emission spectra for the stains are shown in Figures 1 and 2. The spectra were obtained from samples of the Vybrant DyeCycle stain bound to DNA. The Vybrant DyeCycle Green stain/DNA complex has fluorescence excitation and emission maxima of 506/534 nm, respectively. The Vybrant DyeCycle Orange stain/DNA complex has fluorescence excitation and emission maxima of 519/563 nm, respectively.
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Figure 1. Fluorescence excitation and emission spectra for the Vybrant DyeCycle Green stain bound to DNA in TBE,pH 8.3. | Figure 2. Fluorescence excitation and emission spectra for the Vybrant DyeCycle Orange stain bound to DNA in TBE,pH 8.3. |
Table 1. Contents and storage information.
Material | Amount | Concentration | Storage* | Stability | |
---|---|---|---|---|---|
Vybrant DyeCycle Green stain Vybrant DyeCycle Orange stain | 400 μL | 5 mM solution in dimethyl sulfoxide (DMSO) |
| When stored as directed, this kit is stable for at least 6 months. |
Number of assays: Sufficient material is supplied for approximately 200 fl ow cytometry assays based on a 1 mL test volume.
Approximate fluorescence excitation/emission maxima: Vybrant DyeCycle Green stain: 506/534 nm, bound to DNA; Vybrant DyeCycle Orange stain: 519/563 nm, bound to DNA.
Materials Required but Not Provided
Caution
The hazards posed by these stains have not been fully investigated. Since Vybrant DyeCycle Green and Orange stains are known to bind to nucleic acids, treat the stain as a potential mutagen and use with appropriate care. The stains are 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. These stains can also be used for cells suspended in Hanks’ Balanced Salt Solution (HBSS) or phosphate-buffered saline (PBS). 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 a Vybrant DyeCycle 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. The Vybrant DyeCycle 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 Staining Protocol
This basic protocol is optimized using Jurkat cells suspended in complete medium (RPMI/10% fetal bovine serum) and stained with Vybrant DyeCycle stain at 37˚C.
![]() | Figure 3. Histogram of live Jurkat cells stained with Vybrant DyeCycle Green stain showing DNA content distribution. G0/G1 and G2/M phase histogram peaks are separated by the S-phase distribution. Excitation at 488 nm was used with a 530/30 bandpass filter. |
A.![]() | B.![]() |
Figure 4. Histogram of live Jurkat cells stained with Vybrant DyeCycle Orange stain showing DNA content distribution. G0/G1 and G2/M phase histogram peaks are separated by the S-phase distribution. Panel A shows the distribution of this population of cells when 488 nm excitation was used with a 585/42 bandpass filter. Panel B shows the same population when 532 nm excitation was used with a 585/42 bandpass filter.
MP 35004 2–Feb–2009