FlowJo v9 Manual



FlowJo has several analysis platforms that provide capabilities beyond the gating and statistics used for most simple data analysis. All analysis platforms are fully integrated into the FlowJo paradigm: i.e., they can be easily applied to multiple samples (usually, by using Groups), they are easy to edit, and all operations are saved with the Workspace for your permanent record.

Workspaces and Demo Data are provided for several of FlowJo's platforms so that you can try out these features yourself (Tutorial downloads page). In addition, we have several lessons that give step-by-step instructions for FlowJo platforms and features (FlowJo University page).

FlowJo has the following analysis platforms:


Kinetics analyses provide you a powerful tool for computing parameter statistics as a function of time and exporting the data or graphics, or statistics computed as a function of time.� If you did not collect a time parameter, FlowJo will add one for you.� You can even use the Layout Editor to overlay time-series graphs to compare different samples.

Cell Cycle Analysis

FlowJo gives you a sophisticated, yet easy-to-use interface to model DNA/Cell cycle data.� You can simultaneously compute several different standard models, with the ability to constrain different model parameters with the click of a mouse.

Proliferation Studies

FlowJo models proliferation data obtained using cell tracking dyes such as CFSE. FlowJo presents a graphical display as well as information about each generation in the subset. The proliferation platform also provides information about the fraction of cells from the original population that have divided, and the number of times these cells have divided. In addition the FlowJo Proliferation Platform draws gates that separate each generation.

Population Comparison

FlowJo can take two or more samples and analyze them to determine how different the distributions are. Comparisons can be univariate or multivariate, and you can rank multiple samples by their similarity to controls.


Using this platform, you can calibrate any of your fluorescence parameters.� The platform can use a calibrated bead set as a standard, a stained sample as a reference, or numbers that you enter to define the calibration manually.� FlowJo creates a calibrated parameter and displays all graphs and statistics in calibrated units such as absolute number of molecules per cell.


Sometimes your data is not properly compensated... sometimes, it is not possible to compensate your data on the machine because of the complexity of the analysis.� FlowJo's compensation platform makes compensating data much easier than compensation on the machine; you are always guaranteed a correctly-compensated output!

Derived Parameters

Let FlowJo calculate a ratio of two parameters, add a time parameter (in case you want to do Kinetics analyses), or convert between log and linear scaling.

Boolean Gates

You can combine data from existing subpopulations into a gate with your choice of Boolean properties: And, Or, And Not, Or Not.


An analysis platform unique to FlowJo:� view your data dynamically.� Use the Movie Platform to generate graphs as a function of time (kinetic analyses), or to generate a graph of one or two parameters as a function of a third.� This unique visualization lets you uncover subtle relationships in your data that would be impossible to see otherwise.

Sample Validity

FCS files can suffer from flaws introduced by the cytometer during acquisition. FlowJo can test the validity of selected samples in the Workspace, or of all samples as they are loaded into the workspace. You can adjust the sensitivity of the test to allow small measurement fluctuations. You can exclude parameters that are not pertinent to the current analysis.

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