You are sitting in a seminar when you glimpse it: the figure that beats all other figures – a beautiful contour plot – and you realise that a similar figure is what you need to publish your paper in a high-ranking journal. You know the basics of flow cytometry, but admit you need a little help to design the experiment.
Lucky for you, there are a lot of different resources available online to help you out. A quick internet search can help you find simple tools for designing your experiments, whole online tutorials, tips for panel design or lead you to courses you can attend.
To help you plow through the many choices, I’ve covered some of the more useful online resources here.
Go to the Source for Help
Most of the companies that sell flow cytometers and/or reagents have a host of information on their websites.
Firstly, look at reputable company websites and well-known core facilities for information. For example the Life Technologies website has some excellent tutorials on the basics behind fluorescence and flow cytometry.
Other companies offer webinars about aspects of flow cytometry. Often the webinars are given by experts in the field and are recorded so you can view them at your leisure. It is worth adding yourself to the mailing list of big companies so you are aware of events they are hosting or advertising. You never know, you might get better discounts and even freebies if you are signed up to their mailing lists.
Choosing your Fluorochromes: Spectral Viewers
The choice of your fluorochrome is crucial to your flow cytometry experiment. One of the most useful online tools, spectral viewers, can help you find the right dyes for your needs.
Spectral viewers allow you to easily look at the excitation and emission of fluorochromes. You can use spectral viewers to view the spectra of all of your dyes, determine spectral overlap between your dyes, and look at alternative dye combinations to minimise compensation in your experiment. In addition, spectral viewers can help you determine which filter/channel you should use to collect the emitted light. Some spectral viewers, such as the one on the BD Biosciences website, also have the standard configuration of their machines preloaded making the process easier.
The Life Technologies spectral viewer even comes as a handy App for your iPhone, so if you desperately need to know which filter to use for DAPI when you’re at the pub, you have the answer instantly.
One word of caution: spectral viewers on company websites tend to only display products that their company markets, so it is worth looking at other spectral viewers to get the spectra for all of your dyes. And if your dye isn’t available on a spectral viewer, a Google search should reveal the excitation and emission spectra.
Putting it Together: Product Search and Panel Design
There are multitudes of antibodies/cell stains available for flow cytometry. It can be an overwhelming task to find the right panel of antibodies for your experiment. Several resources are available online to help with this.
A good resource for finding antibodies is Chromocyte (www.chromocyte.com), which offers a “free impartial resource for flow cytometry, antibody search and cell based assay products, knowledge and services”. The Chromocyte website helps you design your antibody panel and search all the catalogues of major antibody manufacturers for antibodies. The website also has other resources including a mailing list, advertising of meetings, some educational resources and job listings.
A similar website is Fluorish from Treestar, which helps with panel design and has a database of antibody catalogues from major manufacturers.
Doing the Deed: Online Protocols
It can be quite daunting when you need to prepare and stain a cell/tissue type for the first time, however there are many resources available online that allow you to search for protocols. Many antibody companies have basic staining protocols online, core facility websites often provide their tried and true protocols, and protocols can be gleaned from online articles..
When deciding on a protocol, read several of them and don’t be afraid to contact the person who wrote the protocol to ask for assistance.
The Experts: Purdue Cytometry List
The Purdue Cytometry List (http://www.cyto.purdue.edu/hmarchiv/index.htm) was established in 1989 and has over 4000 members, many of whom regularly contribute to the list. These members range from scientists, to core facility staff, cytometrists with decades of experience as well as some of the founders of the technique. This mailing list is a fantastic resource for any questions regarding cytometry and the list archive is great for looking up past questions and reading responses from the world leaders in cytometry.
It is free to join, however just to warn you, the list is very popular and can see over 10 questions/answers being shared a day, so your inbox can fill up quickly. It is worth searching the archive for your question before posting on the list as it is likely that your topic has been covered before.
So the next time you are planning the quintessential flow cytometry experiment, be sure to make use of these online tools – they may just turn a good experiment into a great one!