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 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). 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 (Picture 1). Vybrant® DyeCycle™ Orange stain is excited using both 488 nm and 532 nm laser lines with emission ~570 nm (Picture 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 Pictures 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.
Picture 1. Fluorescence excitation and emission spectra for the Vybrant® DyeCycle™ Green stain bound to DNA in TBE,pH 8.3.
Picture 2. Fluorescence excitation and emission spectra for the Vybrant® DyeCycle™ Orange stain bound to DNA in TBE,pH 8.3.
主要试剂
Vybrant® DyeCycle™ Green or Orange Stain
Cells and culture medium
主要设备
Flow cytometer tubes
实验步骤
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.
1. Remove the Vybrant® DyeCycle™ Green stain or Vybrant® DyeCycle™ Orange stain from the freezer and allow it to equilibrate to room temperature.
2. Prepare flow cytometry tubes each containing 1 mL of cell suspension in complete media at a concentration of 1 × 106 cells/mL.
3. To each tube add 2 μL of Vybrant® DyeCycle™ Green stain or Vybrant® DyeCycle™ Orange stain. Final stain concentration is 10 μM. After use, seal the stain vial tightly. The stain in DMSO solution may be subjected to many freeze-thaw cycles without reagent degradation.
4. Incubate at 37˚C for 30 minutes, protected from light.
5. For cells stained with Vybrant® DyeCycle™ Green stain, analyze samples on flow cytometer using 488 nm excitation and green emission (Picture 3). For cells stained with Vybrant® DyeCycle™ Orange stain, analyze samples on a flow cytometer using 488 nm excitation or 532 nm excitation and orange emission (Picture 4).
Picture 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.
Picture 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. The picture shows the distribution of this population of cells when 488 nm excitation was used with a 585/42 bandpass filter.
注意事项
For optimal DNA content cell cycle analysis, follow these guidelines:
1) Eliminate cell clumps and aggregates from the cell suspension before staining
2) Use 37˚C for incubation with the Vybrant® DyeCycle™ stains, keep cells at 37˚C until acquisition
3) Staining may be performed at room temperature, with the staining time about twice as long as the 37˚C incubation time
4) Do not use glass containers with this stain
5) Do not wash or fix cells after staining cells with Vybrant® DyeCycle™ stains
6) Validate flow cytometry instrument performance on the day of use
7) Use linear amplification for DNA content
8) Use low flow rate for acquisition
9) Collect adequate numbers of events for the intended application
10) 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
11) Eliminate or correct for cell aggregates during data analysis using gating or modeling software
附 件 (共4个附件,占22KB)
Picture 2.gif
5KB 查看
Picture 3.gif
6KB 查看
Picture 1.gif
5KB 查看
Picture 4.gif
6KB 查看