When FlowJo 2.1 was first released in June 1997 it generated considerable interest in our laboratory. For some time we had been frustrated with the applications available for analyzing our flow cytometric data and FlowJo promised to overcome many of those frustrations. Three years, several program revisions, many experiments and thousands of megabytes of data later, FlowJo has proven to be an extremely valuable tool. We use the current version 3.2 (released July 2000) every day to analyze and present the data from our highly complex flow cytometry-based experiments.
FlowJo is based around “workspace” documents that contain the samples from your experiment, along with all of the gates, graphs, statistics, tables and reports that you apply to, or generate from, your data. This means that you have a complete and persistent record of all the analysis you perform, enabling you to easily review what you have done or to leave an analysis and return to it later. The workspace approach also makes batch analysis extremely quick and simple as samples can be placed into groups and then the same analysis applied to all the samples with a single drag ‘n drop. When new samples are added to the workspace FlowJo can be setup to automatically match them to pre-existing groups and automatically perform the same analysis and reports.
On top of the workspace FlowJo adds a comprehensive set of data presentation tools that include all the standard flow cytometric plots, as well as some extremely useful new types, in particular probability contour plots with outliers. Excellent publication quality output can be generated directly in FlowJo or transferred to other applications for further manipulation. As I read the literature I can often pick the authors who use FlowJo from the quality of their flow cytometric data presentation. Furthermore, statistics from a large number of samples can be quickly generated, collated and exported for further analysis in Excel or other specialized packages. These two features alone make FlowJo worth the price to us. In addition FlowJo has a number of other advanced capabilities, including software compensation and cell-cycle and kinetics analysis platforms. This is all topped off with excellent support from the authors and an active development cycle that sees new features added and bugs fixed on a regular basis.
It is hard to find negative aspects to the current version of FlowJo, partly because the authors have been extremely responsive to user suggestions. In the past I have been hesitant to recommend previous versions as they were somewhat unstable, but the current version is a major improvement in that regard (if you were put off in a previous version by instability then it is worth another look). Some people may not like the fact that the manual is available only online as a series of web-pages. However, this does allow the authors to keep the manual up to date with changes in the program and nearly every window in FlowJo has a direct link to the manual making it simple to find help when you need it (the web-pages can be download in their entirety if you have a slow or non-permanent internet connection). Perhaps the biggest complaint I have heard about FlowJo is that is can be difficult to learn how to use, especially if you have been trained on other flow cytometric software. This is not due to any major problems in the design or layout of the program, but rather in adjusting your thought patterns and workflow to best make use of FlowJo features (a helpful tutorial is available). FlowJo can require some effort initially and sometime I wish for a little more flexibility but I have found that FlowJo encourages a structure to my thinking and planning that enforces a consistent analysis style that is well worth the cost.
For some people the fact that FlowJo is available only for the Macintosh will be the major drawback. However, while FlowJo may not be quite compelling enough to justify buying a Mac just to run it, I think it comes extremely close! FlowJo 3.2 requires a PowerPC running System 7.5 or greater, with a least 16MB of RAM. However, if you are analyzing data of any size or complexity I would recommend a G3 or G4 with plenty of RAM. FlowJo can read FCS files from any instrument, collected on an HP, PC or Mac. A 60-day trial version is available.
In summary, FlowJo is an extremely powerful program that does its job uncommonly well. We have not seen a better package for flow cytometry analysis and presentation and it is has become indispensable in our lab. If you are serious about flow cytometry then FlowJo deserves careful consideration.
Adrian Smith
PhD Student
T Cell Biology
Centenary Institute of Cancer Medicine and Cell Biology
Sydney, Australia