Vybrant®DyeCycle™Rubystain

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 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 cell populations is then used to produce a frequency  histogram that reveals 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™ Ruby stain from Invitrogen offers  near-infrared emission for DNA content analysis in living cells. The  Vybrant® DyeCycle™ Ruby stain is cell membranepermeant, DNA-selective,  and essentially nonfluorescent until bound to double-stranded DNA. This  stain 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. Vybrant® DyeCycle™ Ruby stain may  also be used with any excitation source from 488 nm through to 690 nm  wavelengths. Staining protocol is simple; the suspended cells are  incubated in the presence of Vybrant® DyeCycle™ Ruby stain and  fluorescence is measured directly—no additional treatment or  centrifugation is required. The Vybrant® DyeCycle™ Ruby stain allows  simultaneous co-staining of the cell population for other parameters and  offers the possibility of cell sorting based on DNA content.

The fluorescence excitation and emission spectra of the stain are  shown in Figure 1 and were obtained from samples of the dye bound to  DNA. The Vybrant® DyeCycle™ Ruby stain/ DNA complex has fluorescence  excitation and emission maxima of 638 nm and 686 nm, respectively.

实验材料

Contents and storage information.

Number of assays: Sufficient material is supplied for 400 assays  (Cat. no. V10273) or 100 assays (Cat. no. V10309) based on the protocol  below.

Approximate fluorescence excitation/emission maxima: Vybrant® DyeCycle™ Ruby stain: 638/686 in nm, bound to DNA.

Materials Required but Not Provided

• Cells and culture medium or Hanks’ Balanced Salt Solution (HBSS)

• Flow cytometry tubes

Caution

No data are available addressing the mutagenicity or toxicity of  Vybrant® DyeCycle™ Ruby stain. Since Vybrant® DyeCycle™ Ruby stain binds  to nucleic acids, treat the stain as a potential mutagen and use with  appropriate care. Handle the DMSO dye solution with particular caution  as DMSO is known to facilitate the entry of organic molecules into  tissues. Always wear suitable protective clothing, gloves, and eye/face  protection when handling this reagent. Dispose of the reagent 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. Growth  medium or buffer used, cell density, cell type variations, and other  factors may influence staining. In initial experiments, try a range of  stain concentrations to determine the optimal stain concentration for  the given cell type, buffer, and experimental conditions. 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  significantdifferences in fluorescence signal. If Vybrant® DyeCycle™  Ruby stain is used in combination with other dyes for multicolor  applications, apply other dyes to the sample first following  manufacturers’ instructions, including washes. Apply the Vybrant®  DyeCycle™ Ruby stain as the last stain 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,  and eliminate or correct for cell aggregates during analysis using  gating or modeling software

• Staining with Vybrant® DyeCycle™ Ruby stain may be performed at room temperature with the staining time about twice as long

• Hanks’ Balanced Salt Solution (HBSS) is recommended if media is not used, do not use phosphate buffers

• Do not use glass containers with Vybrant® DyeCycle™ Ruby stain

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

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

• 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 intended application

Vybrant® DyeCycle™ Ruby Cell Staining Protocol

This staining protocol is optimized using Jurkat cells suspended in  complete medium (RPMI/10% fetal bovine serum) and stained with Vybrant®  DyeCycle™ Ruby stain at 37˚C.

1. Remove the Vybrant® DyeCycle™ Ruby stain from the freezer and allow the stain to equilibrate to room temperature.

2. Prepare flow cytometry tubes each containing 0.5 mL of cell suspension in complete media at a concentration of 5 × 10⁵ cells/mL.

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

4. Incubate at 37˚C for 15–30 minutes, protected from light.

5. Analyze  without washing cells on a flow cytometer using 488 nm or 633/5 nm  excitation and >670 nm emission (Figure 2). The Vybrant® DyeCycle™  Ruby stain may be excited with any excitation source from 488 nm through  690 nm wavelengths.

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