流式细胞仪检测细胞周期分析：遵循细胞生命周期

niwanmao

国外博客的一篇博文，流式中文网转载并对照翻译如下：

Cell Cycle Analysis by Flow Cytometry: Flowing your Way through Life’s Cycle
流式细胞仪检测细胞周期分析：遵循细胞生命周期

Over the past few decades the mammalian cell cycle has been well documented. Although there are lots of checkpoints as cells move through the cycle, we can very simply divide the cell cycle into three stages according to the DNA content in the nucleus.
在过去的几十年里，哺乳动物细胞周期已经有据可查。虽然在细胞周期进行时存在很多检查点（checkpoints），但我们仍可以很简单地根据细胞核DNA含量将细胞周期分为三个阶段。

When cells are either quiescent or not dividing they have the normal DNA complement (i.e., in normal human cells this is 46 chromosomes worth). When cells initiate proliferation they enter the S (synthetic) phase of the cell cycle and begin replication of DNA. They continue to make new DNA until they double their DNA content at which point they enter mitosis and produce two daughter cells that can either exit the cell cycle or go on to another round of cell division. Simple really!
当细胞是静止的或不分裂，它们有正常的DNA含量（如在正常的人类细胞有46条染色体含量的DNA）。当细胞开始增殖进入S期（合成期）并开始DNA的复制，它们会持续制造新的DNA直至DNA含量翻倍，之后即进入有丝分裂期产生两个子细胞。此时，就可以退出细胞周期或开始另一轮的细胞分裂，产生新的DNA。真的很简单！

Dy(e)ing DNA? No it’s just a pretty color!

We can assess which phase a cell is in by staining the DNA with either a chromogenic or fluorescent dye. Flow cytometry is a perfect technique to quantitate fluorescence and we can use the fact that there is a range of fluorescent dyes that bind to DNA to do this and DNA analysis was one of the first applications of flow cytometry back in the 1960s. DNA analysis by flow cytometry is now used in research applications and in the clinic.
我们可以利用显色剂或荧光染料标记DNA，从而评估细胞位于哪个周期。流式细胞仪是定量荧光最好的方法，而且有较多的荧光染料可以用来做DNA分析。DNA分析是流式细胞仪最早的应用之一，可追溯到上世纪60年代。采用流式细胞术进行DNA分析目前已用于研究和临床应用。

What are the things that need to be considered when designing an experiment to do this?
在设计DNA检测的实验时需要考虑哪些事情呢？

Know your cytometer!
首先，了解您的流式细胞仪！

Although many modern cytometers have multiple laser excitation sources, you need to ensure that it has the right one for the dye you want to use. It also has to have the correct optical detection filter. This leads on to the fact that you need to know about the excitation and emission properties of the dye you want to use. We’ll cover this in other articles.
虽然许多现代流式细胞仪已有多个激光源，但是你仍需明确是否存在能够正确激发DNA染料的激光源。同时还需要存在正确的光学检测滤片。所有这些都是需要你了解染料的激发和发射特性。本文中我们将会覆盖这点。

So, which dye do I use?
然后，我需要用哪种染料呢？

As well as being fluorescent, the dye has to bind stoichiometrically; i.e., in proportion to the amount of DNA that is in the cell. A quick search will reveal a number of these dyes, but they all have different spectral properties.
染料需要能够发出荧光，同时还能够与DNA发生化学结合。目前存在很多种这类染料。

Many are excited by the blue (488nm) laser that is present in virtually all flow cytometers; these dyes include propidium iodide, 7-aminoactinomycin-D and SYTOX® Green.
许多是蓝激光（488nm）激发的，几乎所有流式细胞仪都具备，这些染料包括碘化丙碇（PI）、7-AAD和SYTOX® Green。

Some are excited by other common laser lines such as the red (640nm) laser; examples are TO-PRO-3 iodide and DRAQ5™.
还有一些是由其它常见的激光器如红激光（640nm）激发的，例如TO-PRO-3碘化物和DRAQ5™。

Some, such as the Hoechst dyes require UV laser excitation, which is less commonly available.
其它如Hoechst染料就需要紫外激发，配备较少。

Therefore, knowing which lasers you have in your cytometer will allow you to pick which dye to use.
因此，了解你的流式细胞仪有助于你选择正确的染料。

How do I get the dyes into the cell?
如何使染料进入细胞？

With the majority of DNA binding dyes, cells need to be fixed or permeabilised in order for the dye to enter the cell so they are, in effect, dead. Fixatives used include ethanol, methanol and formaldehyde; cell permeabilising agents include Triton-X100, NP-40 and saponin. Each has its advantages and disadvantages. In general a cleaner profile is seen with the permeabilising methods but these don’t allow long term storage of the samples. Fixatives do allow storage and the fixative of choice is usually 70% ethanol.
多数DNA结合染料都需要细胞预先经过固定或破膜，细胞基本上都是死亡的。固定剂包括乙醇、甲醇和甲醛。细胞破膜剂包括Triton-X100，NP-40和皂素。每种试剂都有其优点和缺点。多数的固定剂均选择70％的乙醇。

I need to keep my cells viable?

Well, now your options are more limited. There are only a few dyes that enter live cells without any fixation or permeabilisation step; namely Hoechst 33342, DRAQ5™ and the DyeCycle dyes from Life Technologies. However, care is needed: optimisation of dye concentration and incubation time is required for every cell system you are using, and be sure to assess cytotoxicity and cell functionality! However, these dyes and precautions will allow you to keep the cells alive in, for example, sorting experiments.
如果你需要保持细胞存活状态，那么就选择很少了。只有少数几种染料可不经过固定或破膜就可以进入细胞，主要是Hoechst 33342、DRAQ5™和DyeCycle系列染料。然而，需要注意的是：需要注意染料浓度和孵育时间上的优化，并注意评估细胞毒性和对细胞功能的影响！

Anything else I need to know?

The cytometer set up is slightly different when running a DNA analysis experiment than when doing a multiple immunophenotyping experiment. With DNA analysis we are in the realm of high resolution data analysis: there is only a doubling of fluorescence between cells in the resting state and those in mitosis so we use a linear amplification to see and display the fluorescence on our cytometer.
DNA含量检测时的流式细胞仪设置与免疫分型略有不同，DNA分析需要高分辨率，需排除细胞粘连的干扰。

We want to make sure that we measure single cells rather than two cells that are stuck together or pass through the laser beam very close together; therefore, removal of cell doublets is important. This can be achieved by analysis of the pulse of fluorescence produced as a cell traverses the laser beam; cell doublets will take longer to pass through the beam than a single cell and have a wider pulse.

The sample is run using a low pressure differential to keep the sample core as small as possible to keep variations to a minimum. This will lead to DNA histograms with low CVs (coefficient of variation) and will allow better discrimination of the cell cycle phases.

Mission accomplished!

So, a little thought about how you optimise your dye to your cytometer, how you prepare the cells and knowing the tricks for running samples for DNA analysis will allow much better experimentation!

原文地址：http://flowcytometry.bitesizebio ... ysis-of-cell-cycle/

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