To help you get a basic understanding of flow cytometry, we offer some introductory explanations and provide you with a list of further resources.
What Is Flow Cytometry?
Flow cytometry uses fluorescent probes, usually labeled antibodies, to identify cells. Cells or particles tagged with fluorescent molecules enter the cytometer via a fluid stream. The molecules then pass by a laser, which emits a specific wavelength of light. The fluorescent signal is detected and amplified, then translated into an electronic signals, which is sent to the computer. Information about the relative size, shape, and fluorescence of cells is recorded and the result is a visual presentation describing the population of cells in question. If needed the cells or particles can be separated by sorting and further analyzed.
What is Cell Sorting?
Cell sorting differs from analysis since it involves the physical separation and isolation of various cell populations. Cells can be sorted using flow cytometry or magnetic labeling to differentiate and separate the cell populations. Cell sorting is similar to standard flow cytometry except that the stream is vibrated at a frequency that separates it into droplets. These droplets are given an electric charge according to their fluorescent profile and flow through an electric field that sends the charged drops of interest into a collection tube or plate while the unwanted cells are directed into a waste container.
How Flow Cytometers Work
This is a relatively complex question; since one can spend hours depending on how deep into the subject you want to go, we suggest some resources that will allow you to dive into the architecture of flow cytometry as far as you wish to go.
- One of the best interactive tutorials on how flow cytometers work is an applet at Becton-Dickenson website. Introduction to Flow Cytometry will give you a clear understanding of how cytometers work
- Another Becton-Dickinson document, Introduction to Flow Cytometry: A Learning Guide , is essential reading if you wish to use our flow cytometry facility.
- Serotec has also produced an excellent education booklet, Introduction to Flow Cytometry.
Flow Cytometry Spectral Analysis Tools
One critical aspect of flow cytometry is for users to understand exactly what is being excited, the excitation spectra and associated emission spectra. This information is critical because the reagents used for fluorescent or confocal microscopy cannot always be used in flow cytometry. Fluorescent and confocal microscopes use mercury arc lamps that emit a number lines of excitation, while the sources used in flow cytometers are coherent single lines of excitation.
Within the last ten years, the number of available fluorochromes has dramatically increased. A fluorochrome (or fluorophore) is a fluorescent chemical compound that can re-emit light upon light excitation. If you are using a new fluorochrome that will be used in the core facility for the first time, you must provide the core director with the excitation and emission profiles so that we can determine the best excitation source and detection for the fluorochrome. Em-max and Ex-max (maximum emission and excitation, respectively) can be a starting point for making this decision, but having the excitation and emission spectra are preferred.
We request users wishing to use new fluorophores to double check these reagents with the Flow Core Director and staff; some reagents suitable for confocal or fluorescent microscopy may not be capable of being excited or detected by the cytometer. Before investing time and money in your experimental design, please ensure that your reagents can be used with our flow cytometers. When in doubt, please consult the flow core director.
We offer two resources to help you gather the specifications of the fluorophore you wish to use.
- On the Becton-Dickenson Spectral Viewer site , you will need to enable the Java applet on your computer; this tool is one of the most handy for helping you determine what reagent can be used in a flow cytometer.
- On the Life Technologies Fluorescence SpectraViewer site , you identify your specific reagent and it will show you both the excitation and emission spectra.
We encourage users when doing more than about 5 to 6 colors to consult FluoroFinder. FluoroFinder is a company that not only projects the antibody companies inventory but also will display the percentage overlap of any given pairs of fluorochromes. This is to aid the user in minimizing the spectral overlap when conducting multicolor panels. FluoroFinder has the base configuration of most of the instruments in the core. Listed currently are the LSRII, Fortessa, and both Aira's Please see the HMRI FluoroFinder link to use this tool.
Resources and Articles
We provide some further resources below to help you learn more about flow cytometry.
Academic, Research and Organization Links
Purdue University offers resources for everything related to flow cytometry, including an email archive for the flow cytometry listserv. The Scripps Research Institute offers a similar library but on a smaller scale. The National Flow Cytometry Resource at Los Alamos National Laboratory focuses on cutting-edge research into improving flow cytometry measurements. The International Society for the Advancement of Cytometry’s (ISAC) mission is to facilitate the exchange of knowledge in the quantitative cell sciences.
For organizational resources, in addition to ISAC mentioned in the previous paragraph, there is the International Clinical Cytometry Society (ISSC), an organization that proactively identifies regulatory, reimbursement and practice challenges affecting the cytometry community’s ability to deliver patient care in all geographies.
A lot of important cytometry knowledge comes from the manufactures who create cytometry tools and devices:
- Beckman Coulter
- Becton Dickinson
- Current Protocols, Wiley and Sons
- DeNovo Software (FCS Express)
- Expert Cytometry
- Molecular Probes, Life Technologies
- Miltenyi Biotec
- Purdue University
- R & D Systems
Houston Methodist Links