流式细胞实验成功的关键：充分的计划和准备
流式细胞术是一种生物学技术，用于对悬浮于流体中的微小颗粒进行计数和分选。这种技术可以用来对流过光学或电子检测器的一个个细胞进行连续的多种参数分析。 流式细胞术（Flow CytoMetry，FCM）是对悬液中的单细胞或其他生物粒子,通过检测标记的荧光信号，实现高速、逐一的细胞定量分析和分选的技术。Flow cytometry is a way to analyze single cells using the scattering properties of light. A cell suspension containing the sample is hydrodynamically focused into a stream of single cells. A laser going through the stream scatters when cells pass through it, and also excites fluorophores attached to the cells. Detectors detect the pattern of light scatter, and also the light emitted from the fluorophores of single cells. Usually detectors are placed in line with the laser beam, and also perpendicular to the laser. This allows better discrimination of signals, for example when using more than one fluorophore whose wavelengths must be distinguished. Today flow cytometers can measure thousands of cells per second – well suited for high throughput studies, screening, or fluorescence activated cell sorting. Though the method sounds straightforward, success will come sooner if you pay attention to some pointers from the bench, regarding potentials pitfalls and surefire prep steps to smooth the way.
首先，学习流式技术基本原理
Powerful though flow cytometry may be, it need not be intimidating. “Don't be scared,” says James Marvin, director of the Flow Cytometry Core Facility at the University of Utah Health Sciences Center. “A four-color assay can easily be run on a four-laser instrument with little background knowledge.” But don’t underestimate the power of talking to seasoned flow cytometry researchers, whether at your nearest core facility, or in a neighboring lab. “A ton of time, money, and tears can be avoided with a simple 1-hour consultation,” says Marvin. “An expert can help select the appropriate reagents and ensure that all the necessary controls are designed for proper data analysis.”
For researchers who are new to flow cytometry, taking some time to learn the basics will pay off quickly. “Knowing a remedial amount about how fluorescence is generated, how fluorescently-tagged antibodies are detected on cells, and how the data are displayed will really get someone far,” says Nancy Fisher, director of the Flow Cytometry Core Facility at the University of North Carolina at Chapel Hill. “There are a number of excellent tutorials on the web offered by instrument manufacturers such as Beckman, BD Biosciences, and Life Technologies that have animated illustrations on how the instrumentation works.”
样品准备：细胞悬液的过滤和细胞浓度鉴定
Today scientists use flow cytometry to analyze cells from many types of body tissues. “Sample prep can make a huge impact on end results,” says Marvin. “In some situations, sample preparation or tissue dissociation methods can make or break the feasibility of a project.” There is a wealth of tissue dissociation products available to prepare samples for flow cytometry. Marvin recommends testing them out, while keeping in mind what’s optimal for your cells, and their possible downstream assays. Because an uninterrupted flow stream is important, Fisher recommends filtering the cell suspension through a 40 or 70 µm nylon mesh right before use. “If using primary tissue, adding DNase and some reducing agents such as DTT or 2-ME [mercaptoethanol] can reduce clumping and mucous,” she says. “Avoid calcium in the media if the cells come from blood, as residual fibrinogen can form clots after the anticoagulant is removed.”
Another crucial preparatory step is determining and using the proper cell concentrations. “It’s amazing how few researchers seem to have any idea how many cells are in each of their tubes,” says Marvin. “Flow cytometers these days are more times than not limited in throughput by volume, and not event rate. Resuspending cells in the appropriate volume can cut your time on the instrument in half, or more.”
恰当的设置实验对照
Designing the proper controls is also a simple but important step, which may involve as many as 5 control tubes for one experimental tube, says Fisher. “Cutting corners on controls is the worst mistake a researcher can make when preparing their experiment,” she says. “By including appropriate controls, set-up time is minimized and their valuable sample is not used up trying to figure out the appropriate instrument settings.”
Don’t forget to re-evaluate those controls when changing your experimental design. Even scientists with some experience doing flow cytometry unwittingly make errors that could have been prevented. “A common problem happens when investigators who are used to running one or two fluorophores start a new experiment adding on fluorophores, or using a different instrument with more lasers,” says Fisher. Instead, “the experiment needs to be re-designed, as adding colors randomly does not necessarily work.”
如何区分活细胞和死细胞？
Analysis tools are important too, such as a method to differentiate between live and dead cells, both of which would scatter light and could still retain some fluorophore labels. “Running live/dead discrimination really cleans up data nicely and avoids artifacts,” says Fisher, referring to the technique of labeling dead cells’ nuclei using a dye such as propidium iodide, which does not enter live, intact cells. The dead, dye-marked cells can be excluded from later data analysis.
总结
Above all, simply paying attention to the fundamentals of the technique will smooth out your flow cytometry work considerably. “Simple steps such as filtering samples to avoid clogs, including appropriate controls, and taking the time to understand how the instrument works will circumvent the vast majority of problems,” says Fisher. Hopefully steps like this will prevent problems that will never have to be solved.
原文链接：http://www.bioku.net/archives/3983
首先，学习流式技术基本原理
Powerful though flow cytometry may be, it need not be intimidating. “Don't be scared,” says James Marvin, director of the Flow Cytometry Core Facility at the University of Utah Health Sciences Center. “A four-color assay can easily be run on a four-laser instrument with little background knowledge.” But don’t underestimate the power of talking to seasoned flow cytometry researchers, whether at your nearest core facility, or in a neighboring lab. “A ton of time, money, and tears can be avoided with a simple 1-hour consultation,” says Marvin. “An expert can help select the appropriate reagents and ensure that all the necessary controls are designed for proper data analysis.”
For researchers who are new to flow cytometry, taking some time to learn the basics will pay off quickly. “Knowing a remedial amount about how fluorescence is generated, how fluorescently-tagged antibodies are detected on cells, and how the data are displayed will really get someone far,” says Nancy Fisher, director of the Flow Cytometry Core Facility at the University of North Carolina at Chapel Hill. “There are a number of excellent tutorials on the web offered by instrument manufacturers such as Beckman, BD Biosciences, and Life Technologies that have animated illustrations on how the instrumentation works.”
样品准备：细胞悬液的过滤和细胞浓度鉴定
Today scientists use flow cytometry to analyze cells from many types of body tissues. “Sample prep can make a huge impact on end results,” says Marvin. “In some situations, sample preparation or tissue dissociation methods can make or break the feasibility of a project.” There is a wealth of tissue dissociation products available to prepare samples for flow cytometry. Marvin recommends testing them out, while keeping in mind what’s optimal for your cells, and their possible downstream assays. Because an uninterrupted flow stream is important, Fisher recommends filtering the cell suspension through a 40 or 70 µm nylon mesh right before use. “If using primary tissue, adding DNase and some reducing agents such as DTT or 2-ME [mercaptoethanol] can reduce clumping and mucous,” she says. “Avoid calcium in the media if the cells come from blood, as residual fibrinogen can form clots after the anticoagulant is removed.”
Another crucial preparatory step is determining and using the proper cell concentrations. “It’s amazing how few researchers seem to have any idea how many cells are in each of their tubes,” says Marvin. “Flow cytometers these days are more times than not limited in throughput by volume, and not event rate. Resuspending cells in the appropriate volume can cut your time on the instrument in half, or more.”
恰当的设置实验对照
Designing the proper controls is also a simple but important step, which may involve as many as 5 control tubes for one experimental tube, says Fisher. “Cutting corners on controls is the worst mistake a researcher can make when preparing their experiment,” she says. “By including appropriate controls, set-up time is minimized and their valuable sample is not used up trying to figure out the appropriate instrument settings.”
Don’t forget to re-evaluate those controls when changing your experimental design. Even scientists with some experience doing flow cytometry unwittingly make errors that could have been prevented. “A common problem happens when investigators who are used to running one or two fluorophores start a new experiment adding on fluorophores, or using a different instrument with more lasers,” says Fisher. Instead, “the experiment needs to be re-designed, as adding colors randomly does not necessarily work.”
如何区分活细胞和死细胞？
Analysis tools are important too, such as a method to differentiate between live and dead cells, both of which would scatter light and could still retain some fluorophore labels. “Running live/dead discrimination really cleans up data nicely and avoids artifacts,” says Fisher, referring to the technique of labeling dead cells’ nuclei using a dye such as propidium iodide, which does not enter live, intact cells. The dead, dye-marked cells can be excluded from later data analysis.
总结
Above all, simply paying attention to the fundamentals of the technique will smooth out your flow cytometry work considerably. “Simple steps such as filtering samples to avoid clogs, including appropriate controls, and taking the time to understand how the instrument works will circumvent the vast majority of problems,” says Fisher. Hopefully steps like this will prevent problems that will never have to be solved.
原文链接：http://www.bioku.net/archives/3983
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