Revised: 12–January–2009 | MP 10347
FxCycle™ Violet Stain
Catalog no. F10347
Introduction
Analysis of nucleic acids is a common application of flow cytometry. Measurement of DNA
content allows the study of cell populations in various phases of the cell cycle as well as
analysis of DNA ploidy. 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 stains that
exhibit emission signals proportional to the DNA mass. Flow cytometric analysis of these
stained populations is then used to produce a frequency histogram that reveals the various
cell cycle phases.
Univariate DNA content analysis is an established assay method and is widely used for studies
in oncology, cell biology, and molecular biology. Using flow cytometry, multicolor cell cycle
studies are possible, and it is advantageous to analyze DNA content on alternative lasers to
preserve the common 488 nm laser for other markers. Well suited for the popular violet laser
line, FxCycle™ Violet stain (4', 6-diamidino-2-phenylindole, dihydrochloride) can also be used
with UV excitation. With DNA content measurement on the violet laser, other parameters
such as cyclins, cyclin-dependent kinases, cell cycle checkpoints, nuclear proteins, and
proliferation markers can be measured on the familiar 488 nm laser. FxCycle™ Violet stain
preferentially stains dsDNA; it appears to associate with AT clusters in the minor groove.5
Binding of FxCycle™ Violet stain to dsDNA produces a ~20-fold fluorescence enhancement.6
For long-term storage, the dye is supplied as five vials of 100 μg solid dye and stored at 2–6˚C;
individual vial stock solution in deionized water is also stored at 2–6˚C for convenience.
Table 1. Contents and storage information.
Material Amount Storage* Stability
FxCycle™ Violet stain 5 × 100 μg
2–6˚C
Desiccate
Protect from light
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•
•
When stored as directed the product is
stable for at least 1 year.
Number of assays: Sufficient material is supplied for 500 assays based on the protocol below.
Approximate fluorescence excitation/emission maxima: FxCycle™ Violet stain: 358/461 nm, bound to DNA.
FxCycle™ Violet Stain | 2
Before Starting
Materials Required but Not
Provided Deionized water
Reagents for fixing cells such as alcohol or formaldehyde
Reagents for permeabilizing cells such as Triton® X-100
Buffer such as phosphate buffered saline (PBS)
Caution The hazards posed by FxCycle™ Violet stain has not been fully investigated. The stain is a
known mutagen and may cause sensitization by inhalation and skin contact, and is irritating
to eyes, respiratory system, and skin. Do not breath dust. In case of contact with eyes, rinse
immediately with plenty of water and seek medical advice. Wear suitable protective clothing,
safety glasses, and gloves. Avoid contact with skin and eyes. Dispose of the reagents in
compliance with all pertaining local regulations.
Preparing Stock Solution To make a 1 mg/mL stock solution of FxCycle™ Violet stain, add 100 μL deionized water to
one vial of the stain. Mix well. Store this solution at 2–6˚C, protected from light. When
stored as directed, this FxCycle™ Violet stock solution is stable for at least six months.
Spectral Characteristics The fluorescence excitation and emission spectra of the FxCycle™ Violet stain are shown
in Figure 1. The spectra were obtained from samples of the stain bound to DNA with
fluorescence excitation and emission maxima of 358/461 nm respectively. FxCycle™
Violet stain may be used with the violet 405 nm excitation laser commonly found on flow
cytometers, as well as UV excitation.
•
•
•
•
Wavelength (nm)
Fluorescenceexcitation
Fluorescenceemission
300 550500350 400 450 600
Figure 1. Fluorescence excitation and emission spectra of FxCycle™ Violet stain bound to dsDNA.
FxCycle™ Violet Stain | 3
Experimental Protocol
The following staining procedure was developed using the Jurkat T-cell leukemia cell line,
but can be adapted for any cell type. Fixative, permeabilization reagent, cell density, cell type
variations, and other factors may influence staining.
In initial experiments, try a range of stain concentrations to determine the concentration
that yields optimal staining for the given cell type and experimental conditions. All fixative
should be removed from cells before proceeding with cell staining, however staining with
FxCycle™ Violet stain may be done concurrent with the addition of a permeabilization reagent
if desired. 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 FxCycle™ 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. FxCycle™ Violet stain should be the last stain applied to the sample, and do not
wash samples prior to flow cytometric analysis.
General Guidelines For optimal DNA content cell cycle analysis, follow these general guidelines:
Eliminate cell clumps and aggregates from the cell suspension before staining
Validate flow cytometry instrument performance on the day of use
Use linear amplification for DNA content
Use low flow rate for acquisition
Collect adequate number of events for the intended application
Cells must be fixed before staining with FxCycle™ Violet stain for DNA content cell cycle
Do not wash cells after staining with FxCycle™ Violet stain
Staining Procedure
1.1 Harvest the cell sample(s).
1.2 Fix cells according to your preferred protocol.
1.3 Wash cells. Remove all fixative from cells before proceeding with the cell staining.
1.4 Using an appropriate buffer, adjust the sample cell concentration to be 1 × 106
cells/mL.
1.5 Prepare flow cytometry samples each containing 1 mL cell suspension. Optional
permeabilization reagent may be added.
1.6 Add 1 μL of FxCycle™ Violet stain to each flow cytometry sample and mix well.
1.7 Incubate flow cytometry tubes for 30 minutes at room temperature or 2–6°C, protected
from light.
1.8 Analyze samples without washing in a flow cytometer, using 405 nm excitation and emission
collected in a 450/50 bandpass or equivalent.
Example of results obtained using FxCycle™ Violet stain for DNA content cell cycle analysis is
shown in Figure 2.
•
•
•
•
•
•
•
FxCycle™ Violet Stain | 4
References
1. Current Protocols in Cytometry, 7.0.1–7.27.7 (2004); 2. Practical Flow Cytometry, 4th Ed., Shapiro HM, Ed. (2003); 3. Methods Mol Biol 281, 301
(2004); 4. Cytometry A 58, 21 (2004); 5. Biochemistry 26, 4545 (1987); 6. Biochem Biophys Res Commun 170, 270 (1990).
Figure 2. Histogram of HL-60 promyeloblast cells stained with FxCycle™ Violet stain showing DNA content distribution.
HL-60 cells were fixed overnight with alcohol, washed, and then resuspended in 0.1% Triton® X-100/PBS/1% BSA before
staining with FxCycle™ Violet stain for 30 minutes at room temperature. G0/G1 and G2/M phase histogram peaks are
separated by the S-phase distribution. Analysis was performed at 405 nm excitation with a 450/50 bandpass filter.
50 100 150 200 250
(x 1,000)
0
500
1,000
2,000
1,500
Numberofcells
FxCycle™ Violet fluorescence
Product List Current prices may be obtained from our website or from our Customer Service Department.
Cat. no. Product Name Unit Size
F10347 FxCycle™ Violet Stain *for flow cytometry* *500 assays* *DAPI* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 set
F10348 FxCycle™ Far Red Stain *for flow cytometry* *500 assays*. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 set
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L23101 LIVE/DEAD® Fixable Green Dead Cell Stain Kit *for 488 nm excitation* *200 assays*. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 kit
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GAS-003 Fixation and Permeabilization, 1 × 5 ml . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 tests
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GAS002S-100 Permeabilization Medium - Bulk, (MEDIUM B), 1 × 100 ml. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1000 tests
10010-049 Phosphate Buffered Saline (PBS) 7.2 (1X), liquid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 × 500 mL
20012-050 Phosphate Buffered Saline (PBS) 7.4 (1X), liquid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 × 500 mL
14025-092 Hanks’Balanced Salt Solution (HBSS) (1X), liquid, contains calcium and magnesium, but no phenol red . . . . . . . . . . . . . . . . . . . . . . . . . . . . 500 mL
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12091-039 RNAse A (20 mg/ml). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 mL
FxCycle™ Violet Stain | 5
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