PerspectivesAre you interested in submitting a Perspective Article? Be sure to read The Science Advisory Board's Editorial Guides for Perspective Articles. Click here. Flow Cytometry: A Brief Introduction and Guide to On-Line Resources by Beverly E. Barton, Ph.D. If a picture is worth a thousand words, what are thousands of pictures worth? This statement might sum up the power of flow cytometry, a technique which gives us the ability to measure fluores-cence and scatter parameters of individual cells. Flow cytometers use lasers to scatter light from particles (cells, bacteria, chromosomes, etc.) and to activate fluorescent dyes coupled to tagging molecules (usually antibodies). Software creates histograms and compares or quantifies fluores-cence and/or scatter data. In practical terms, the limit for the number of parameters (two scatter di-rections, forward and perpendicular, and fluorescence parameters) is not availability of fluorochromes or lasers, but software to resolve and display data. Flow cytometry has become a popular technique for phenotyping, cell cycle analysis, apoptosis assays, assessing gene expression, calcium flux measurements, and much more. Our laboratory uses it in place of western blot analysis, because analytes can be quantified on an individual cell ba-sis, making it particularly useful for mixed cell populations. We also use it to assess transfection efficiencies, using either a fluorescent oligonucleotide or a plasmid containing the gene for one of the green fluorescent proteins. Mostly, we use it to quantify apoptosis in our cancer studies. Flow cytometry started as a simple instrument designed during World War II to detect bacteria or spores in aerosols for defense against germ warfare. Instead of a liquid sheath fluid, this instrument used air to visualize particles. In 1953, P.J. Crossland-Taylor published a description of liquid sheath fluid for particle enumeration (Nature 171: 37-38; 1953). In 1965, Mark Fulwyler at the Los Alamos National Laboratory built an instrument that sorted based on volume; in 1971 Louis Kamentsky at Columbia’s IBM-Watson Laboratory built a sorter that used a syringe pump to extract the selected population. Fluorescence detection was added in the late 1960’s, which improved analyses. By now investigators used flow cytometry to analyze cell cycle kinetics, aneuploidy of tumors, and cellular DNA content. Kamenetsky sent one of his early sorters to Leonard Herzenberg at Stanford in the 1970’s, who used an argon laser (in place of an arc lamp) to activate the fluorochromes on antibodies directed against cell-surface markers and to sort cells based on their fluorescence intensities. The technology was sold to Becton-Dickinson in 1974, and the fluorescence-activated cell sorter (FACS) was born. The range of resources available on the internet for novice and experienced flow users is truly amazing. I’ve grouped resources by topic and indicated whether one needs a paid subscription for access. General Flow Cytometry ResourcesPurdue University Cytometry Laboratory Website Start learning about flow cytometry on the web at this site. The most comprehensive flow cytometry and analytical cytology website I have found. You will find resources ranging from archived protocols to request forms for the latest protocols CD-ROMs - and they are free while supplies last. Job listings, meetings, journals, latest developments- it is all here. Lectures Website Dr. J. Paul Robinson, Purdue University School of Veterinary Medicine, has put a compilation of lec-ture slides on a website. Some of the information is bullet statements, but the pictures are great. If you’ve never seen a flow cytometer, you’ll see lots of them here. If you are new to flow, you’ll find in-troductory slides here. Practical Flow Cytometry, Fourth Edition by Howard Shapiro, published by J. Wiley & Sons, 2003. Website Dr. Shapiro has kindly put his textbook online through Molecular Probes. It is free! Yes, you do need to register to see it, but that’s all you need to do, and you have access forever. Flow Cytometry E-mail Discussion Group Website Have a question? Novices to experts, all levels of questions are welcome. This e-mail group has been invaluable to me. E-mails are archived and can be searched by topic. International Society for Analytical Cytology Website The premier society for flow cytometry, it publishes Cytometry Parts A and B (Clinical Cytometry), Case Studies in Clinical Flow Cytometry, and Current Protocols in Cytometry. These are available on-line, but you must be subscribed for access. Regular dues are US $126; fees are deeply dis-counted for students, post-doctoral fellows, and scientists from developing nations. Please consider joining. National Flow Cytometry Resource, Los Alamos National Laboratories Website Where the cutting edge happens. NFCR runs a course every June that alternates between Los Ala-mos and New England. I recommend it to the serious flow person; you will learn a tremendous amount, including how to build your own flow cytometer, if you are so inclined. FlowCyte Website This discussion group is dedicated to flow users interested in developing uniform bioinformatics standards. They are also developing analysis and statistics software for flow users. Vendors – Instruments, Lasers, ServiceBecton-Dickinson Immunocytometry Systems Website Owner of the trademarked acronym FACS, BD is perhaps the most well-known manufacturer of flow cytometers and cell sorters, especially for research application. I have one of these instruments. Beckman-Coulter Website Beckman bought Coulter instruments in the past decade to offer a complete line of instruments for clinical and research laboratories. MoFlo Website Now part of Dako, this manufacturer is well-known for its “open” configuration sorters. Its CyAn ana-lyzer acquires up to 11 parameters digitally. Partec Website Headquartered in Germany, Partec has been making flow cytometers since 1969. Their website lists various analyzers and sorters. Apogee Flow Website British company Apogee makes a military flow cytometer, rugged enough for field use for detection of pathogenic bacteria. Guava Technologies Website Guava calls its instruments “personal” flow cytometers, and they certainly are small. Being small allows Guava to claim that their analyses require fewer cells. But flow cytometer software allows you to select the number of cells to analyze per sample, so I am not sure about this. Also, one of the features that makes flow cytometry so powerful is the analysis of thousands of cells per sample. However, if you are on a limited budget, desire a new instrument, and have limited space, these may be perfect for your needs. Luminex Website Their instruments analyze the contents of cell supernatant fluids or lysates through use of antibodies and microspheres. Analytes (up to 100) are measured simultaneously (multiplexing). Luminex makes the instruments; multiplexing kits are available from many antibody vendors. BD and other companies make similar instruments too. Icyt Website ICyt sells lasers and customized cell-sorters. Data acquisition is digital using their proprietary X-Cyte software. Cytek Website Cytek sells used flow cytometers, services flow cytometers, and can upgrade your old flow cytometer. Their pricing for service contracts and for repair is very competitive. I used them to repair the laser on my own instrument. Cytobuoy Website These flow cytometers are made for marine and aquatic use, bench-top and in situ (and on subma-rines). Their flow cells are the optimal sizes for marine algae, bacteria, rather for than mammalian cells like BD, Beckman-Coulter, etc., which have clinical uses as their major markets. Coherent, Inc. Website A major supplier of lasers for flow cytometry and other applications. Their website has a lot of infor-mation about the newest lasers. Newport/ SpectraPhysics Website Another major laser supplier. Evergreen Website Sales and servicing of lasers. Vendors - Fluorochromes, Dyes, etc.Molecular Probes Website Molecular Probes, now part of Invitrogen, has one of the largest selections of fluorescent dyes for a multitude of purposes. Their on-line handbook (also available as a CD-ROM and in print) is a thor-ough resource, not just for flow cytometry, but for all types of fluorescence imaging. Pharmingen Website Part of Becton-Dickinson, Pharmingen has an enormous offering of antibodies. I usually check here first for primary, labeled, and second-step antibodies. Biolegends Website I’ve been finding the prices of antibodies I commonly use very competitive from Biolegends. Caltag Website Owned now by Invitrogen, Caltag offers a wide range of second-step antibodies, and streptavidin conjugates (for use with biotinylated primary antibodies). Spherotech Website Bangs Labs Website Both of these companies make calibration beads for calibration, compensation, and quantification. Free Protocols on the WebUCLA flow cytometry lab protocols Website Good selection of the basics; you’ll have to optimize for your cells, antibodies, etc. in any case. University of Pennsylvania School of Dental Medicine flow lab Website More than just protocols, there are links here, and a simple introduction to flow cytometry. Pharmingen protocols Website These were optimized for Pharmingen products, but really will work with other antibodies and rea-gents. NCI Experimental Therapeutics flow lab Web-site Nice feature on this site: protocols are available as pdfs you can download for your notebook. Baylor College of Medicine flow lab Website Recipes, protocols, links, and more. Software, Free and OtherwiseFree software listed by Purdue Cytometry lab Website Free may be good for you (if you are on a PC, you may like WinMidi). I personally cannot recom-mend any of these because I use Macintosh computers, and most of these were written for Windows or DOS. But I do know many people who like WinMidi (written by Dr. Joe Trotter, when he was at Scripps Institute, La Jolla, CA). SoftFlow, Inc. Website SoftFlow offers two free software packages, FCAP-List and HP to Mac. FCAP-List looks similar to WinMidi in that it will analyze FCS files, but on a Mac rather than a PC; HP to Mac will take files gen-erated on the Hewlett-Packard computer that came with your or your core facility’s flow cyotmeter and convert them to a format readable by software on a Mac. SoftFlow also has free FCS filters for Windows. CytoWin Website This free software package can analyze FCS or ASCII files generated by BD, Beckman-Coulter, or Cytomation (Dako) instruments- check the website for the complete list and for updates. CellQuest Pro Website This software is free with the purchase of a BD instrument (as is ModFit for cell cycle analysis, and BD quality-control software for calibration of the instrument). Separately, this was about US $1000 a couple of years ago. I do like it for acquisition and analysis; its Snap Tool identifies populations for me when I’m viewing density plots, and it has a nice export feature that converts histograms into high-quality jpg files for use in publications and presentations. It does lack the ability to perform post-acquisition compensation, which means that for folks with more than one laser, another software package must be purchased to perform cross-beam compensation. Weasel Website This software is only $200 for a licensed copy, runs on either Windows or Macintosh (native for OS X in version 2), and has many of the same features as more expensive software. I am in the process of trying it out to supplement CellQuest Pro. Summit v.4 Website Dako software designed for the CyAn and the MoFlo to acquire and analyze data. Uses Windows XP; contact Dako through website for pricing information. FlowJo Website Treestar’s software runs very well on Macintosh, is reported to be buggy on PCs. I tried it, and found it to be very complicated; its gating logic doesn’t match that of CellQuest Pro, my acquisition software, so I could not use it for some kinds of post-acquisitional analyses. But I know many satisfied users- it is one of the most popular third-party software packages for flow cytometry. This software was US $900 for the Mac version three years ago. WinList Website I personally bought this because I needed to perform post-acquisition spectral compensation for some of my studies (about US $1200). I view it as a necessary software package, but I do not love it. The version for Macintosh runs in Classic and there are no current plans to upgrade to OS X anytime soon (the company offered to buy me a PC and switch my program to the PC version last month; I declined). I suspect they will do nothing now, with the advent of the Intel Macs running Bootcamp (allows Macs to use Windows operating system along with the Mac OS). That said, it does have its many fans. FloMax FCM Website Partec’s proprietary software for Windows offers cross-beam compensation and many other features of FlowJo and WinList. No pricing information was available on the site. Phoenix Flow Systems Website Phoenix offers low-cost software for various types of acquisition and analysis; they occasionally have refurbished instruments for sale. TrainingRealistically, training on an instrument requires hands-on time under the tutelage of an instructor. Learning to analyze data after acquisition is a matter of understanding flow cytometry and learning a software package. All instrument vendors offer training usually in conjunction with instrument pur-chase or through regularly-schedule courses. BD Immunocytometry web-based training Website If you have access to a BD instrument, and someone to help you out, this site is a good place to get started learning to acquire and analyze stained samples. Mario Roederer’s Spectral Compensation Site Website This is an excellent place to teach yourself more about spectral compensation if you are compensating before acquiring your data. FloCyte Website FloCyte offers regional flow cytometry training at a reasonable cost; schedules and syllabuses of courses are listed. ### << Previous Next >> [ View All Perspectives ] |
|
