Vybrant® DyeCycle™ Violet Stain

实验概要

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). 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.

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.

实验材料

Contents and storage information.

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

• Cells and culture medium

• Flow cytometer tubes

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.

实验步骤

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:

• Eliminate cell clumps and aggregates from the cell suspension before staining

• Use 37˚C for incubation with the Vybrant® DyeCycle™ Violet stain

• Hanks’ Balanced Salt Solution (HBSS) is recommended if media is not desired, however phosphate buffers are not recommended

• Do not use glass containers with this stain

• Do not wash or fix cells after staining cells with Vybrant® DyeCycle™ Violet stain

• Validate flow cytometry instrument performance on the day of use

• Use linear amplification for DNA content

• Use low flow rate for acquisition

• Collect adequate numbers of events for the intended application

• Eliminate  dead cells from the DNA content analysis of living cells using a dead  cell discriminating stains such as SYTOX® Green, SYTOX® Red or SYTOX®  AADvanced™ dead cell stains or LIVE/DEAD® Fixable Dead cell stains such  as Green, Red, Far Red, or Near-IR kits

• Compensation may be required for Alexa Fluor® 488 and PE channels

• Eliminate or correct for cell aggregates during data analysis using gating or modeling software

• Human  and rodent stem cells efflux Vybrant® DyeCycle™ Violet stain, and this  is the basis for the Side Population (SP) technique; the efflux can be  blocked with verapamil, fumitermorgin C, or other such blocking agents,  to prevent dye efflux for accurate DNA content analysis in these stem  cells

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.

1. Remove the Vybrant® DyeCycle™ Violet stain from the refrigerator and allow the vial to equilibrate to room temperature.

2. Prepare flow cytometry tubes each containing 1 mL of cell suspension in complete media at a cell concentration of 1 × 10⁶ cells/mL.

3. To each tube, add 1 μL of Vybrant® DyeCycle™ Violet stain and mix well. Final stain concentration is 5 μM.

4. Incubate at 37˚C for 30 minutes, protected from light. Keep cells at 37˚C until acquisition.

5. Analyze  samples without washing or fixing on a flow cytometer using ~405 nm  excitation and ~440 nm emission (Figure 2). Vybrant® DyeCycle™ Violet  stain may also be excited with a UV light source.

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