General Info Obtaining FlowJo Compatibility Error Messages Upgrading to Version 6 Getting Started Getting Help Sample File Annotation Graphs Histograms Gating Graphical Layouts Tables of Statistics

Advanced Data Analysis Groups Preferences Exporting Graphics, Statistics and FCS files Report Generation Tricks and Tips Platforms Compensation Display Transformation of Compensated/Digital Data Kinetics Derived Parameters Cell Cycle Proliferation Population Comparison

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Getting Started
2.1 General Help
  2.1.1 How do I get started?
  2.1.2 How do I get help?
2.2 Sample File Annotation
  2.2.1 My data files were not correctly labeled during collection. Can FlowJo fix the parameter labeling?
  2.2.2 What are FCS file Keywords?
  2.2.3 What is ProJo? How much is it? Where do I get it?
  2.2.4 Why do all my data files have the same name in the Workspace window?
  2.2.5 FlowJo sorts my data files incorrectly...
  2.2.6 How do I change the order of my samples in a Workspace, Table or Layout?
2.3 Graphs
  2.3.1 Where can I find information explaining FlowJo Graphs?
  2.3.2 What are all these different graph types? What are they useful for?
  2.3.3 Graph gates and frequencies don't appear in the Layout Editor or upon export.
  2.3.4 When I display a dot plot, not all the events appear in the graph. Why?
2.4 Histograms
  2.4.1 How do I get more information about Histograms?
  2.4.2 Can I create gates on Histograms?
  2.4.3 I want to see an overlay of different histograms at the same time. How do I do that?
  2.4.4 What does the % of Max stands for on the Y axis of overlayed histograms?
  2.4.5 How do I change the presentation of histograms (color of lines, offset)?
  2.4.6 How do you get statistics for an overlay plot in the Layout Editor?
2.5 Gating
  2.5.1 Sometimes when I alter gates, their frequencies don't show up in the workspace, other times they do. Why? How do I get the frequency to show up?
  2.5.2 I am trying to select a gate that is entirely underneath another gate, but when I click I just select the topmost gate.
  2.5.3 What is a node in the Workspace window?
2.6 Graphical Layouts
  2.6.1 Where do I find information explaining the Layout Editor?
  2.6.2 How are Layouts useful in my analysis? What sorts of things can I use Layouts for?
  2.6.3 I want all of my graphs to be added to a Layout at a scaling other than 100%?
  2.6.4 How do I copy Layouts from one workspace to another?
  2.6.5 How do I make a Layout that includes sample statistics?
2.7 Tables of Statistics
  2.7.1 Where do I find information explaining the Table Editor?
  2.7.2 I created a table and multiple columns have identical values.
  2.7.3 How do I reuse my analysis when looking at the next set of samples?
  2.7.4 I created a table and multiple cells in the table are blank. Why?

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Advanced Data Analysis
3.1 My workspace window is very cluttered with gates and analyses-it takes me forever to scroll to the sample I want. How can I clean it up all at once?
3.2 How can I make repeatedly analyzing similar experiments easier?
3.3 In the Layout Editor, I don't have "Placeholders" selected, yet I get a placeholder for a graphic. When I generate the batch output, I don't see any graphs for that item.
3.4 How do I make movies of my data?
3.5 Groups
  3.5.1 How do I create Groups
  3.5.3 I have adjusted the gates on some samples in a group and now I want to change them back?
3.6 Preferences
  3.6.1 What do all the different preferences do?
  3.6.2 Why do the preferences change on different computers?
  3.6.3 Why does FlowJo always ask me to save my files, and how do I get it to stop?
3.7 Exporting Graphics, Statistics and FCS files
  3.7.1 What can I export using FlowJo?
  3.7.2 How can I transfer my data to a drawing program or a spreadsheet program?
  3.7.3 Can I produce publication quality graphics in FlowJo?
  3.7.4 What file types are the graphics exported as?
  3.7.5 When do I need high resolution?
  3.7.6 When I paste graphics into Canvas 7, all the graphs turn purple! Help!
  3.7.7 Exporting outliers to Illustrator.
  3.7.8 How can I transfer my data to a Statistics program?
  3.7.9 Where can I get clip2gif?
3.8 Report Generation
  3.8.1 What is iteration?
3.9 Tricks and Tips
  3.9.1 Using the Option Key.
  3.9.2 Can I drag analyses from Workspace to Workspace?
  3.9.3 I organize my data in folders. Can I add these folders directly to FlowJo? How will I keep all this data organized in the Workspace?

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Platforms
4.1 Compensation
  4.1.1 I need to compensate my data. I know the compensation percentages. Can I enter them directly, instead of running singly stained samples and letting FlowJo derive the compensation matrix?
  4.1.2 How do I edit the compensation matrix?
  4.1.3 How do I apply the same compensation matrix to all of my workspaces?
  4.1.4 Do I only need one singly stained compensation control tube per color?
  4.1.5 What can I do if my data was overcompensated during collection?
  4.1.6 How can I make compensated data that is squished against the axis look better?
  4.1.7 Where can I get more information about compensation?
4.2 Kinetics
  4.2.1 How can I determine the fraction of responding cells?
  4.2.2 I have determined that only 40% of my cells respond; how do I display the fluorescence of only the responding cells?
  4.2.3 I wish to perform more complex statistical analyses on the kinetics data using other programs. How can I get the kinetics data?
  4.2.4 What if I didn't collect time as a parameter?
  4.2.5 How do I derive a ratio of Ca++ fluorescence?
  4.2.6 How do I determine the Ca++ concentration instead of the fluorescence ratio?
  4.2.7 How is time divided to calculate the kinetics functions?
4.3 Derived Parameters
  4.3.1 What is the formula for Gain and Offset?
4.4 Cell Cycle
  4.4.1 How do I remove doublets and cell fragments from my analysis?
  4.4.2 Why doesn't G1, S and G2 add up to 100%?
  4.4.3 How does FlowJo choose the gates to create the G1, S and G2 subpopulations?
4.5 Proliferation
  4.5.1 Why don't the numbers from the model and gates match?
  4.5.2 What do the division index, proliferation index, and percent divided mean?
  4.5.3 What does the RMS tell me about the model fit?
4.6 Population Comparison
  4.6.1 What do the different statistics tell me for univariate comparison?
  4.6.2 How do I determine the biologically meaningful T(X)?
  4.6.3 What is the appropriate number of events to collect for multiparameter population comparison?
  4.6.4 What number of bins should be used?
4.7 Calibration

 General Info [top]

1.1 How can I learn about FlowJo?
FlowJo software is used for the analysis of flow cytometry data. We provide a number of support mechanisms to help you get started including two Tutorials (Basic and Advanced), Application Tech Notes (Getting Started, Compensation, Kinetics), and a complete online Reference Manual (press HELP in any FlowJo window to access the Reference Manual directly). Everything you need to use FlowJo is on our web site so click on the links above or take a look at the web pages describing the program's Features, System Requirements, and how to Contact us (questions are always welcome!).
1.2 What features does FlowJo have that other analysis programs do not?
FlowJo is like the Swiss army knife of Flow Cytometric analysis programs. It contains tools for all of the jobs you need to perform any type of data analysis. Keeping track of your analyses is easy with the Workspace window. FlowJo provides you with publication-quality graphics and tables with very little effort. You can design graphical and tabular layouts as simple or as complex as you wish, with complete control over the fonts, colors, and styles of the graphs. With a single click, you can copy the outputs to your favorite spreadsheet or graphics package--where you can ungroup all of the elements for individual editing. One of the most significant features of FlowJo is its unique and powerful Batch Analysis capability. Propagating any analysis (gates, statistics, platforms) is as easy as drag-and-drop: to other samples, or to entire experiments! FlowJo can apply these intelligently--only to samples that were similarly stained or any given subset of the samples in your experiment. Finally, you can ensure that all samples have exactly the same analysis, or you have the flexibility to have sample-specific modifications of certain gates without affecting batch analysis capabilities. In addition to handling immunophenotyping data analysis with ease, FlowJo also has special data analysis platforms: Cell Cycle, Kinetics (Calcium flux), Proliferation (CFSE), Statistical Comparison of samples, Calibration (Quantitation), and Compensation. In addition, we have added two major new features in Version 4 - the clustering platform finds subpopulations of cells automatically and the multigraph overlay platform allows you to display subsets in different colors while looking at several different parameters.
1.3 Does Tree Star offer training?
We provide FlowJo seminars free of charge as well as in-depth computer training workshops for a fee. Check out our Calendar to see if we will be visiting your area in the future. If you are interested in hosting a Seminar or scheduling a training session, please contact us.

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1.4 Obtaining FlowJo:
1.4.1 How do I evaluate FlowJo? Is there a demo version I can try?
There are two ways to try the fully functional version of FlowJo before buying. When you first launch FlowJo it is working in Demo Mode and it will only read demo data files provided by Tree Star. This is a good way to try the Tutorials.
Evaluation Mode allows you to analyze your own data for free for 30 days. Simply fill out the online form and a serial number will immediately be emailed to you. You can download the current version from the web or request a CD be mailed to you.
1.4.1.1 I'm running the demo the sample files won't open!
Older versions of the program sometimes has trouble reading the demo files. This has been fixed. Download the current version of FlowJo.
1.4.1.2 I downloaded FlowJo and it did not Un stuff itself correctly!
Sometimes the Stuffed folder that contains FlowJo does not open properly. Double click on the icon (flowjo.sea.hqx) to repeat the process. If that doesn't work, download the file again or let us know and we will send you a CD.
1.4.2 Can I try out FlowJo with my own data before I buy?
Absolutely! What better way to find out how powerful this software is, than with your own files? First, download FlowJo. Second, fill out this online form to receive your Evaluation License 30 day Serial Number that activates a fully functional version of FlowJo.
1.4.3 How much does it cost? How do I get it cheaper than that?
Current pricing information about FlowJo is here. Basically, the first license of FlowJo costs $1895 with a 30% academic discount ($1325). There is a discount for purchases of multiple FlowJo licenses when they are ordered on a single PO.
1.4.4 How do I order and pay for FlowJo?
You can order FlowJo online. Click here for more purchasing information. We accept Purchase Orders, credit cards, and checks. Send them to :
Tree Star, Inc.
340 A Street BD1 #203
Ashland, OR 97520

1.4.5 Which License protection method should I choose?
There are three FlowJo license protection options; serial number, dongle and site license. The advantages and disadvantages of each are outlined on the License Options web page.
1.4.5.1 What the heck is a dongle!?
A dongle is a small security device that plugs into your computer so you can run FlowJo. The advantage of the dongle based license is that you can install FlowJo on many computers, and move the dongle around. Warning: moving the dongle will greatly increase the chance that it gets lost, and we do not replace dongles.
1.4.5.2 ADB or USB dongle?
Dongles come in ADB and USB flavors. Older computers (Macs with the old style gray case) usually have ADB ports. The current generation of computers (iMac, blue G3, G4) have USB ports. If you wish to use dongles on both types of computers, there is a third party adapter made by Griffin Technologies.
NOTE: FlowJo Version 4 only runs with a new USB dongle. This dongle is not the same as the version 3 dongles. When you upgrade from version 3, we will send you the new dongle (click here for more info on upgrading).
1.4.6 How come my USB dongle is not recognized by FlowJo?
Version 3: There is an extension that needs to be installed for the USB dongle to work. If you look on the CD that came with your USB dongle, you will find the extension as well as instructions in the Installation Instructions folder. Specifically, copy the MachaspusbDD extension into your extensions folder and then restart your computer. You can also download the USB dongle extension here.
Version 4: You need to run the V4 USB installer. If you look on the CD that came with your new USB dongle, you will find the VISE installer as well as instructions in the Installation Instructions folder. Double click to launch the installer. You can also download the USB dongle installer here.
1.4.7 Do I need a new extension for my USB dongle to work with system OS X/9.2?
Version 3: If you have a USB dongle and have upgraded to OS X/9.2, you will need a new extension/driver for your dongle to work. Download the new USB dongle extension here. Copy the MachaspusbDD extension into your extensions folder and then restart your computer.
Version 4: The version 4 USB dongle needs to be installed. Run the V4 USB installer to install the correct extensions. If you look on the CD that came with your new USB dongle, you will find the VISE installer as well as instructions in the Installation Instructions folder. Double click to launch the installer. You can also download the USB dongle installer here.
1.4.8 Can I upgrade from my current version? How?
All updates (e.g., Version 4.0 to 4.1) are free. Simply download the current version and run it! Version 2.x to 3.x (or from 3.x to 4.x) is considered an UPGRADE and costs $199 (Upgrading from version 2.x to 4.x is $299). Click here to link to our Upgrade page for more details and to determine if your license qualifies for a free upgrade (within a year of purchase). To check the history of changes to the program, visit this page.
1.4.9 I have FlowJo documents from version X, will they still work if I upgrade to version Y?
Yes, FlowJo is "backwards compatible". Any version will read the workspace documents written by previous versions. Note that this is not true for the reverse. Workspaces created by the current version of FlowJo cannot be read by previous version of FlowJo.
1.5.0 I have an ADB dongle and have a new computer with a USB port. Can I exchange the dongle?
Version 3: The cost for exchanging a dongle is $50 plus shipping charges. If you don't want to exchange the dongle, there is an adapter made by Griffin Technology.
Version 4: If you wish to upgrade to version 4 the cost is $199 + shipping (dongle cost is included). Version 4 does not work with ABD dongles.

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1.5 Compatibility
1.5.1 What types of computers does FlowJo run on?
Currently FlowJo will run on the Mac OS platform.
1.5.3 I have files that were collected using Lysis on an HP, CellQuest on a Mac or Expo on a PC. Can FlowJo read them?
FlowJo is guaranteed to read any legal FCS file, versions 1, 2 or 3. It will also read most of the illegal FCS files produced by many programs. FCS files are the same format, regardless of the platform where it was collected.
1.5.4 What about OS X compatibility ? What about Windows compatibility?
Version 3.6.1 will run under OS X (in the "classic" environment). As of Version 4 (released 5/1/02), FlowJo is fully carbonized to run in OSX. The PC/Windows version is under development.

1.6 Error Messages
1.6.1 What should I do when I experience a FlowJo Error message.
Please let us know when you encounter a problem with FlowJo by filling in a Bug report. We appreciate your taking the time to let us know how we can make FlowJo better. It is useful if you let us know what you were doing when you experienced the problem and also what the error message stated (if possible). If the bug is reproducible, then please save a copy of the workspace and the data required to regenerate the bug, and send it to us (contact us for more details). We will strive to fix the bug as quickly as possible!
1.6.2 FlowJo crashes at start up (on computers running less than OS 8.5).
Older versions of the Mac operating system (<OS8.5) do not support "live window resizing". To turn off this feature, launch FlowJo, open the Preferences (under the Edit menu). Under the General tab, check the box next to "Don't use live window operations".
1.6.3 Why FlowJo doesn't find its dongle after I've run CellQuest?
This occurs only with ADB dongles. Once you run CellQuest something happens to the ADB bus, and FlowJo can't see the dongle any more. If you run FlowJo first, (or restart after using CellQuest) then FlowJo will work fine. Another solution is to run the two programs on different computers. But if you frequently switch back and forth between the two programs, and want them on the same computer, you should exchange your dongle for a serial number or USB dongle to resolve the conflict.
1.6.4 Getting your USB dongle to work with OS X/9.2.
Version 3: If you have a USB dongle and have upgraded to OS X/9.2, you will need a new extension/driver for your dongle to work. Download the new USB dongle extension here. Copy the MachaspusbDD extension into your extensions folder and then restart your computer.
Version 4: Run the V4 USB installer to install the correct extensions. If you look on the CD that came with your new USB dongle, you will find the VISE installer as well as instructions in the Installation Instructions folder. Double click to launch the installer. You can also download the USB dongle installer here.
1.6.5 My workspace has become corrupted after a crash, can I recover it?
If this happens, you can hold down the option key, and you'll see that in the File menu the item called Open Workspace will become Recover Workspace. That should allow you to open those workspace. The layouts will be lost, but your gating and samples will be available.

1.7 Upgrading to Version 4
1.7.1 Why should I upgrade to Version 4? How much does it cost?
Version 4 is OSX carbon and has many new features. Upgrading from version 3 costs $199 (from version 2 costs $299). If you have purchased your copy of FlowJo within a year - the upgrade is free. Please visit our upgrade page for more information on upgrading.
1.7.2 Do I have to upgrade to Version 4?
No.
1.7.3 What license protection options are available with Version 4? What operating systems does it run on?
Version 4 runs on OSX (carbon) or any OS 8.6 or newer. You can use either a serial number or a new Version 4 USB dongle for license protection. Note: ADB dongles do not work with version 4.
1.7.4 How do I exchange my version 3 dongle for the new Version 4 dongle?
See our web site for instructions on upgrading and exchanging the dongles. Order the upgrade (either via the web or by contacting us) and we will send you instructions for exchanging the dongles.
1.7.5 Why are all the dialog boxes blank in version 4?
Version 4 of FlowJo requires a Carbon Lib file 1.5 or greater for it to run in OS8 or 9. You can download a new Carbon Lib file from here. Replace the old file in your Extensions folder and restart your computer.

 Getting Started Analyzing Data [top]

2.1 General Help
2.1.1 How do I get started?
There are several ways to get started using FlowJo. We have a Getting Started Tech Note that is a four page guide to analyzing your data with FlowJo. If you would like a more detailed introduction, try the Basic or Advanced Tutorials. In addition, there is an on-line reference manual that can always be accessed from within any FlowJo window by pressing the HELP button.
2.1.2 How do I get help?
We provide a number of support mechanisms including two Tutorials (Basic and Advanced), Application Tech Notes (Getting Started, Compensation, Kinetics), and a complete online Reference Manual (press HELP in any FlowJo window to access the Reference Manual directly). Please contact us with your questions!

2.2 Sample File Annotation
2.2.1 My data files were not correctly labeled during collection. Can FlowJo fix the parameter labeling?
There are two different ways to annotate your data files post collection. Within FlowJo, you can double-click the parameter you would like to change (in the Workspace window) and type in the correct annotation. If the parameter you wish to alter is not displayed as a column in the Workspace, you can Edit Workspace Columns (under the Workspace menu) and add the columns that correspond to the fluorescence channels (or to any other FCS keyword). In the Workspace window you can then double click on the spreadsheet cell (i.e., the under the $P3S column across from the sample name) and type in the correct antibody.
The other option is ProJo - a free FCS file editor, also made by Tree Star. ProJo reads keywords in FCS files, and displays them in an easy interface allowing you to modify their values. ProJo is particularly adept at handling group operations that alter parameters from all of the files in the group. The primary difference between FlowJo and ProJo is that ProJo makes changes in the FCS files themselves, while FlowJo does not.
2.2.2 What are FCS file Keywords?
Sample data files that are collected on flow cytometers all follow an international standard called FCS. Each data file contains all the numeric fluorescence information for each cell, as well as a list of keyword values. FCS keywords organize information such as the date, parameter names (i.e., FL1), parameter stains (i.e., CD8 FITC) etc. Click here for a list of frequently used keywords. The full FCS file format specification can be found here.
2.2.3 What is ProJo? How much does it cost? Where do I get it?
ProJo is a free FCS keyword editor. It opens and displays FCS file(s) and allows you to change keyword values. Original Keywords are renamed and saved within the original file, therefore, you can always revert back to the original parameters. For more information, please visit the ProJo web page, or download ProJo.
2.2.4 Why do all my data files have the same name in the Workspace window?
CellQuest does not write the full name of the data file in $FIL keyword. They leave off the .001 suffix, leaving all the values of the $FIL keyword the same. To deal with this, FlowJo has a Sample Identification preference to "Use data file name". Open the Preferences (under the FlowJo menu) and check the box next to "Use data file name".
2.2.5 FlowJo sorts my files incorrectly, when I try by file name, the files aren't in order!
FlowJo sorts the files in the Workspace window in alphabetic/numeric order based on the name of the sample. You can click the "Sort" button in the Workspace window to sort on another parameter. Or, you can change a Preference so that FlowJo will display each sample's filename instead of the name entered in the $FIL keyword. In most cases the filename and the $FIL keyword value are the same, but some programs do not write the full name of the data file in $FIL keyword. They leave off the .001 suffix, leaving all the values of the $FIL keyword the same. To deal with this, FlowJo has a Sample Identification preference to "Use data file name". Open the Preferences (under the FlowJo menu) and check the box next to "Use data file name".
Note in version 4.0, FlowJo has a new preference allowing you to specify the "name" column to be any (combination of) keywords... even ones you create.
2.2.6 How do I change the order of my samples in a Workspace, Table or Layout?
Layouts and tables are always created in the same order that the samples are in the Workspace. You can reorder by doing a "sort". To display the samples in the order they were collected in sort on the $BTIM keyword (time the sample was collected). To get a specific, desired order, create a new keyword (for example, "Sort order"), and type in the numerical position number for each sample, then sort on that keyword.

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2.3 Graphs
2.3.1 Where can I find information explaining FlowJo Graphs?
To get more information about graphs and data display in FlowJo, visit the reference manual pages describing graphs.
2.3.2 What are all these different graph types? What are they useful for?
FlowJo can display your data in many different plots. Each graph type has advantages and disadvantages. Dot Plots display rare events well; however, these plots frequently saturate in areas of high cell density. Visual information about the cell population in the saturated area of the plot is lost. (Dot plots are drawn showing only 8000 cells by default by FlowJo in order to offset this problem). Contour lines drawn on Contour Plots are similar to altitude contours on geographical maps. Five percent of cells fall between each contour line in a 5% contour plot. One drawback of contour plots is that 5% of cells fall outside of the last contour line and information about rare populations is not visible. In order to not lose information about these populations, click on the “Show Outliers” box and click Apply. Outlier Plots combine the density estimation of contours with the rare event information of a Dot plot. Density Plots provide density information (i.e., the number of events within an area) by using different shades of gray. Zebra Plots are a mix of contour (20%) and density plots. Pseudocolor Dot Plots provide benefits of a dot plot without the loss of information due to saturation. Saturating is avoided by drawing dots in different colors according to the cell density in that area. Turn on the smoothing option for the pseudocolor plot to display a Color Density Plot.
2.3.3 Graph gates and frequencies do not show up in the Layout Editor or when I export the graphs.
There is a preference to "include gates drawn on plot". To change this preference, open the Preferences (under the FlowJo menu), click on the General tab, and check the box next to "include gates drawn on plot". In order to include/remove gates and frequencies from a graph in the Layout Editor, double click item in layout, go to Annotate tab, and enable "Show Gates" and "Show Gate %".
2.3.4 When I display a dot plot, not all the events appear in the graph. Why?
FlowJo displays 8000 events for dot plots by default. This is to prevent saturation in areas of large numbers of cells. You can change this setting in the Preferences (to show a different number or percentage of events). Go under the FlowJo menu and choose Preferences. Click on the "Graphs and Gates" tab and at the bottom left is a sections for Dot Plots. Change the number from 8000 to zero - to show all events. Click Save.

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2.4 Histograms
2.4.1 How do I get more information about Histograms?
To get more information about graphs and data display in FlowJo, visit the reference manual pages describing histograms.
2.4.2 Can I create gates on Histograms?
Yes. While in a Histogram window, simply click and drag the mouse to create a gate. You can alter the gate by either dragging one of the handles or by dragging the entire gate.
2.4.3 I want to see an overlay of different histograms at the same time. How do I do that?
The Layout Editor supports overlays of both univariate (histograms) and bivariate (dot plots) graph types. If you drag a node (sample or subpopulation name) from the Workspace window onto an existing graph in the Layout Editor, FlowJo will overlay the two samples. You can overlay as many graphs as you wish! The order of layers in an overlay can be altered by dragging the rows of the legend into the desired order. To delete a layer from an overlay, hold the option key and click on that row in the legend.
2.4.4 What does the % of Max stand for on the Y axis of overlayed histograms?
When graphs are overlaid on the layout editor and the y-axis is left on automatic scaling, the Y-axis on the overlay histogram is "% of Max" as opposed to "# Cells or events". If you have different numbers of events collected for the two overlaid samples, the histogram for one would be a lot taller than the other. To depict the data in a "normalized" fashion, we use % of Maximum. The % of Max is the number of cells in each bin divided by the number of cells in the bin that contains the largest number of cells.
To make a histogram we take all of the events and divide them into a number of "bins" which are numerical ranges for the parameter on the X axis. Generally, FlowJo uses 256 bins, but that is specific to our program and arbitrary. The number of cells in any given bin will vary if you change the number of bins. Each graph is scaled to the percentage of its maximum bin. If it is important to compare relative numbers of events, then you may wish to have a fixed Y-axis scale. Double click the overlay graph and choose Fixed Scaling under the Specify tab.
2.4.5 How do I change the presentation of histograms (color of lines, offset)?
To change color, click on the colored box in the legend that is next to the name of the sample you want to change and a palette will pop up. Pick the color you like from the palette. To change the line, click on the line next to the color box in the legend and choose from the popup menu. If you wish to change the color and line of a single histogram, first display the legend. Double-click on the histogram and under the Annotate tab, click the Show Legend box. For more information, click here.
Note that version 4 changes all this--you can directly specify all colors for any graph.
2.4.6 How do you get statistics for an overlay plot in the Layout Editor?
Overlays only display the statistic for the sample or subset that is at the top of the legend (simply drag nodes up or down in the legend to change the order). To display the statistics from each sample in an overlay, drag the stats from Workspace. Hint: select more than one statistic in at once by holding the Apple key. Once you drag them to the Layout Editor, they will all end up in one text box.

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2.5 Gating
2.5.1 Sometimes when I alter gates, their frequencies don't recalculate up in the Workspace, other times they do. Why? How do I get the frequency to show up?
There is a Preference that specifies whether re computations should always occur immediately, only if the sample is loaded in memory, or only when needed to create a table or layout. Open the Preferences (under the FlowJo menu) and click the Workspaces tab to choose which method bests suits your analysis needs. If you have large numbers of files or large files, it may not be optimal to have FlowJo recalculate the statistics every time you alter a group's gate. If the file is in memory, the computation of statistics is performed very quickly, but for files which haven't been read into memory yet, or were dumped from memory to make room for other data files, the computation may take longer.
Note that FlowJo will NEVER show you a wrong value. If a gate changes such that a statistic needs to be recomputed, and your preference is "lazy" recalculation, then FlowJo simply shows a blank in the workspace. However, whenever you create a table or layout, FlowJo will compute the statistic so that it is present for export or printing.
You can force FlowJo to immediately calculate gates and statistics. Simply select the node(s) you wanted updated, and select "Recalculate" from the Workspace menu (cmd "="). FlowJo computes the statistics for the nodes you have selected (and any of their children). You can also select a group, and then the recalculate command forces all samples to re compute.
2.5.2 I am trying to select a gate that is entirely underneath another gate, but when I click I just select the topmost gate.
Press the command key while clicking to select gates: this will iterate through all gates in the same area as the mouse, each time selecting the next-lower one. This also works in the Layout Editor to select "hidden" items.
2.5.3 What is a node in the Workspace window?
A node is a line of text in the Workspace window that is written underneath a sample name. A node represents a gated subpopulation, a statistic, or a special analysis platform such as Cell Cycle or Kinetics. You can drag and drop a node (or several nodes at once) to another sample or subpopulation in the Workspace. In addition, you drag nodes to the Table and Layout Editors to define the statistics and graphics you wish to see in the Tables and Graphical reports.

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2.6 Graphical Layouts
2.6.1 How do I find information explaining the Layout Editor?
To get more information about the Layout Editor as well as getting started on reports and iteration in FlowJo, visit the reference manual pages describing Layouts.
2.6.2 How are Layouts useful in my analysis? What sorts of things can I use Layouts for?
The Layout Editor creates graphical reports of your data. There are several report formats to choose from - web pages, movies, or graphics laid out on a page. The main feature of the Layout Editor is its ability to compile a set of graphs/statistics/text etc. from many samples automatically. FlowJo takes a layout you have created from a single sample and then batch processes through all the samples in an experiment to compile the same layout from all the samples.
2.6.3 I want all of my graphs to be added to a Layout at a scaling other than 100%?
For the purpose of fitting more than two graphs per page, it's better to scale the page, not the graphs. There are several ways to scale the page in the Layout Editor. First, zoom out so that you are viewing several pages (denoted by gray lines). Roll your mouse over the intersection of the vertical and horizontal page lines and drag the page to the size you wish. Another useful way to change the page size, is the "scale to page" pull down option at the bottom of the Layout Editor window. This option will scale the page in order to fit all graphs on a single page. The final method to change the page size is to click the "Setup" button and enter a percentage to scale the page.
2.6.4 How do I copy Layouts from one Workspace to another?
Currently there is no option to do that, but we may include it in future versions. The best way is to create a Template Workspace. Once you have analyzed an experiment, you can save the Workspace as a Template (under the File menu) - this saves all of your analyses, groups, table definitions and layouts, but removes the samples from the Workspace. The next time you need to analyze a similar experiment, simply add the new samples to the Template Workspace and most of the work is done for you!
2.6.5 How do I make a Layout that includes sample statistics?
Simply drag the statistics nodes from the Workspace window to the Layout Editor. They will show up in the Layout Editor in a text box. Be aware that if you wish to have the "Frequency of Parent" statistic show up in the Layout Editor, you cannot drag the subset node from the Workspace as this will turn into a graph in the Layout Editor. Instead, add the "Frequency of Parent" statistic to the subset node and then drag the statistic node (represented by sigma icon) .

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2.7 Tables of Statistics
2.7.1 Where do I find information explaining the Table Editor?
To get more information about making tables in FlowJo, visit the reference manual pages describing the Table Editor.
2.7.2 I created table and multiple columns have identical values.
FlowJo creates one column for every statistic that you drag into the Table Editor. So if you drag the same statistic from three different samples into the Table Editor, then each one of those three will be applied to all samples - resulting in three identical columns in the output.
2.7.3 How do I reuse my analysis when looking at the next set of samples?
The best way is to create a Template Workspace. Once you have analyzed an experiment, you can save the Workspace as a Template (under the File menu) - this saves all of your analyses, groups, table definitions and layouts, but removes the samples from the Workspace. The next time you need to analyze a similar experiment, simply add the new samples to the Template Workspace and most of the work is done for you!
2.7.4 I created a table and multiple cells in the table are blank. Why?
Check the Group of samples that is selected. If a statistic is requested for a population that is not defined on a particular sample, FlowJo will leave a blank cell in the table. This can occur when you accidentally select the wrong group of samples, or have not copied populations to all of the samples in a group.

 Advanced Data Analysis [top]

3.1 My workspace window is very cluttered with gates and analyses-it takes me forever to scroll to the sample I want. How can I clean it up all at once?
Click on one of the triangles next to a sample name in the Workspace window and this will close that gating tree. To close all the disclosure triangles on all your samples, hold the Apple key and close one triangle. This will close up that triangle as well all other triangles at the same level.
3.2 How can I make repeatedly analyzing similar experiments easier?
FlowJo provides the capability to create templates of your analyses. Once you have analyzed an experiment, you can save the Workspace as a Template (under the File menu) - this saves all of your analyses, groups, table definitions and layouts, but removes the samples from the Workspace. The next time you need to analyze a similar experiment, simply add the new samples to the Template Workspace and most of the work is done for you!
3.3 In the Layout Editor, I don't have "Placeholders" selected, yet I get a placeholder for a graphic. When I generate the batch output, I don't see any graphs for that item.
The reason that FlowJo places a Placeholder (a box with an X through it) instead of a graph in the Layout Editor is that while batch processing to compile the new layout, FlowJo could not identify a subset corresponding to the original graphic. This is likely due to a discrepancy in the annotation of the samples (i.e., "CD28Green" vs. "CD28FITC"). Note that, by default, FlowJo requires you to match parameters AND stains in order to create a graphic. In other words, each sample must have the same parameters such as "P1", "P2", etc., that are being displayed in the original graph as well as the same stain name for each parameter (such as "CD28", "CD4", etc.). There is an option under preferences which relieves the latter constraint by ignoring the stain name in identifying subsets. Go to Preferences (under the FlowJo menu) and under the Layouts and Tables tab, check the option called "Allow Stain Name Mismatch".
3.4 How can I make a movie of my data?
Animation of successive views of a graph can be an effective mechanism for picking out changes or evolution in cell distributions. There are two ways to make movies of your data in FlowJo. The first type of movie is made from the Layout Editor and each frame in the movie is a different sample. Click here for more information. The second type of movie is generated within a single sample. A bivariate plot is displayed showing only the events that fall within a slice of a third parameter for each frame of the movie. The third parameter can also be time - allowing you to view a bivariate graph as a function of time during collection of the sample. Click here for more information.

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3.5 Groups
3.5.1 Creating groups
To get more information about creating Groups of samples in FlowJo, visit the reference manual page describing Groups.
3.5.3 I have adjusted the gates on some of my samples in a group and now I want to change them back?
Select the gate name(s) that you wish to revert to the original gate that was applied to the group and select "Unify Analyses" from the Workspace menu. If you have selected a gate name from an individual sample, only that sample will have the gate reverted to the original gate. However, if you select the gate name from under the Group, all the samples in the group will revert that gate to the original.

3.6 Preferences
3.6.1 What do all the different preferences do?
Visit this page for a detailed explanation of FlowJo Preferences.
3.6.2 Why do the preferences change on different computers?
Each computer saves a FlowJo Preferences file in its System folder and the Preferences are stored on each computer based on user input. Thus if you go from one machine to another, you may find certain options are different from the machine you're used to.
Note for FlowJo version 4, each user (if the computer is used as multi user with log in) will have their own preferences.
 3.6.3 Why does FlowJo always ask me to save my files, and how do I get it to stop?
The "always use the same answer" box in the Save dialog box only enables that action for this session. When you re launch FlowJo after quitting, you will need to answer the questions once again. Check if there is a Preference for turning off the question entirely. For example, if you do not wish to have FlowJo auto save, you will need to check "always use the same answer" and press No when FlowJo asks you to save at the beginning of a new FlowJo session. To avoid this question, open the Preferences (under the FlowJo menu) and under the Workspaces tab, turn off Auto saving.

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3.7 Exporting Graphics, Statistics and FCS files
3.7.1 What can I export using FlowJo?
You can export graphs, statistics, and even FCS files using FlowJo. For more information, visit the reference manual page describing Exporting.
3.7.2 How can I transfer my data to a drawing program?
Once you have created a graphical layout in FlowJo, it is very easy to transfer this data to a drawing program. A simple way is to copy and paste any graphic from FlowJo to the drawing program. The Layout Editor also has a button (in the top right of the window) for saving the layout to the clipboard or as a file. The most efficient way to transfer a layout is to click the "Save and launch application" button in the Layout editor. This saves the layout as a file, launches your favorite drawing program, and opens the layout in this program.
3.7.3 Can I produce publication quality graphics in FlowJo?
Yes, FlowJo does export publication quality graphics into other programs. You can start learning about this option by reading this.
3.7.4 What file types are the graphics exported as?
FlowJo saves graphics in one of several file formats - GIF, TIFF, JPEG or PICT. PICT is the default format, and is best if you plan to export the graphic to a Macintosh application because you are able to ungroup PICT graphics. The others have varying characteristics are best used when creating web sites or moving data to a Windows PC.
3.7.5 When do I need high resolution?
Use "low resolution" to generate bigger dots in density plots. This option doesn't affect dot plots, and barely affects contour plots. There is also a Preference to draw large dots in dot plots.
3.7.6 When I past graphics into Canvas 7, all the graphs turn purple! Help!
Canvas 7 and several other drawing programs do not read 1 bit drawings. If you turn on the "Export to Canvas 7" Preference (under the General tab), FlowJo will export graphics as 8 bit drawings.
3.7.7 The outliers do not show up when I copy graphs to some drawing programs.
For exporting outliers in plots, we recommend checking the "Export for Canvas 7" Preference. This draws dots as rectangles (8 bit) rather than dots (1 bit), and may result in a better-looking picture.
3.7.8 How can I transfer my data to a Statistics program?
Once you have created a table in FlowJo, it is very easy to transfer this data to a spreadsheet program. Each table has buttons (at the top left) for saving the table to the clipboard or as a file. The most efficient way to transfer a table is to click the "Save and launch application" button. This saves the table as a file, launches your favorite spreadsheet program, and opens the table in this program.
3.7.9 Where can I get clip2gif?
Clip2gif is a program that is required for FlowJo to create JPEG or GIF files. You can find Clip2gif on the FlowJo CD or download it here. Place Clip2gif in the same folder as the FlowJo program.

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3.8 Report Generation
3.8.1 What is iteration?
One of the best things about computers is how they handle repetitive tasks. Show the computer how to do something once, and it will do it a million times without the slightest complaint. In FlowJo's Layout Editor, you can create a layout based on graphs and statistics from one of your samples. With a single click, you can ask it to produce the same layout from all of the rest of your samples.

3.9 Tricks and Tips
3.9.1 Using the Option Key.
The option key is used heavily in FlowJo. Use option dragging to duplicate graphs in the layouts, or option-close to close all your windows at once. Try depressing the option key as you browse through menu items; you will find that many of the menus change to be a different command, giving you quick access to even more powerful features.
3.9.2 Can I drag analyses from Workspace to Workspace?
Yes! Simply open both Workspaces and drag and drop any gates, statistics and platforms you wish.
3.9.3 I organize my data in folders. Can I add these folders directly to FlowJo? How will I keep all this data organized in the Workspace?
You can drag the folders of data to the Workspace window. All the folders of data will be added to the Workspace and their organization is preserved. Any folder is turned into a Group in the Workspace window and the sample files that are contained in that folder are automatically placed in that Group for you.

 Platforms [top]

4.1 Compensation
4.1.1 I need to compensate my data. I know the compensation percentages. Can I enter them directly, instead of running singly stained samples and letting FlowJo derive the compensation matrix?
No, this is not possible. The values used on an instrument are a very different kind of operation than the software uses. Instrument-based compensation may not be correct for more than two colors; FlowJo's compensation is always correct no matter how many colors are used. The percentages you use on an instrument cannot be easily translated to values that the software could use.
4.1.2 How do I edit the compensation matrix?
There is a web page explaining how to edit the file. Editing the matrix file manually is not recommended!
4.1.3 How do I apply the same compensation matrix to all of my workspaces?
FlowJo will allow you to compensate many samples at the same time, simply be selecting all of the desired samples in the workspace, and then selecting the matrix from the Compensation menu. It is good practice to collect a set of compensation controls with each experiment. There are enough day to day variations is laboratory and instrument procedures that require creating a new comp matrix for each experiment. Sometimes you create multiple workspaces for data collected in the same experiment. So the answer is yes, it's possible to apply the matrix to several workspaces--just do a "load compensation matrix" with the file that was saved from the other workspace.
4.1.4 Do I only need one singly stained compensation control tube per color?
Yes.
4.1.5 What can I do if my data was overcompensated during collection?
Unfortunately, overcompensated data cannot be fixed with software compensation.
4.1.6 How can I make compensated data that is squished against the axis look better?
Display the data using a method that has smoothing built into it, such as contour, pseudo-color or density plots. This will spread some of the data in the zero channel out into neighboring space. Note that events smoothed outside of the graph are reflected back into the visible area, so they won't disappear off screen.
4.1.7 Where can I get more information about compensation?
For more information, visit the reference manual page describing Compensation and visit Dr. Mario Roederer's web page explaining compensation.

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4.2 Kinetics
4.2.1 How can I determine the fraction of responding cells?
The fraction of responding cells is defined as the percentage of events with a fluorescence (or ratio) above a certain threshold value. You can set this value manually; choose to display "% of events > threshold" under Function in the kinetics Options box, then click on "Set Threshold." Enter the exact value you want to use as a threshold.
Another strategy might be to define a certain time period of the analysis as the "background" time. This might be the first 30 seconds of collection; perhaps it is the end of the collection period. Create a time slice corresponding to the background period. Then, after clicking on "Set threshold" in the kinetics Options box, select that time slice in the popup menu. Now choose which percentile of the events in that time slice to set as the background. For example, if you choose the 95th percentile, you ask FlowJo to define the threshold as the 95th percentile of events in your time slice (thus, the background time has 5% responding cells). You might also choose the 98th percentile (to limit to 2% responding cells during the background).
The advantage of the second strategy is that when you copy the kinetics analysis to other samples, you don't have to adjust the threshold manually for each sample; rather, FlowJo calculates it with respect to the particular time-period you define as background.
4.2.2 I have determined that only 40% of my cells respond; how do I display the fluorescence of only the responding cells?
Here you have two options. First, realize that the median fluorescence of the top 40% of a population is the 80th percentile of the entire population. Thus, you could display the 80th percentile as a function of time, and you will get a plot that gives you, essentially, the median fluorescence of the responding population as a function of time. Alternatively, you could define a response threshold (see above), and then choose to display the mean or median of events above this threshold.
4.2.3 I wish to perform more complex statistical analyses on the kinetics data using other programs. How can I get the kinetics data?
Simply copy from the graph window and paste into your spreadsheet: you will get two columns. The first column has the time values; the second, the function values displayed in the window. Note that if you have selected smoothing, then smoothed values are copied. In other words, whatever values were used to plot your graph will be copied to the spreadsheet. (Note that there is a Preference that controls whether or not the data values are copied to the clipboard in addition to the graphic; the default setting is to copy both).
4.2.4 What if I didn't collect time as a parameter?
Under the Platforms menu, select Derive Parameters --> "Define new or change". This creates a dialog containing several ways to add new parameters to a data file. One options is the Add Time button. If FlowJo finds keywords in the data file that recorded the start time and end time of the collection session (often recorded by the acquisition software) it will use that to calculate the length of collection. Otherwise you'll be asked to provide the number of seconds to use. When adding the time events, FlowJo will assume a constant rate of collection.
4.2.5 How do I derive a ratio of Ca++ fluorescence?
Under the Platforms menu, select Derive Parameters --> "Define new or change". The first item allows you to add a new parameter that is created from the ratio of two other parameters. Select a numerator and denominator of the ratio, and click the button marked "Add Ratio". When you return to your graphs, you'll see that a new parameter is available in the popup menus.
4.2.6 How do I determine the Ca++ concentration instead of the fluorescence ratio?
The Calcium concentration is not linearly correlated with the ratio of the Indo fluorescence emissions. You need to run a standard to calculate the Calcium concentration.
4.2.7 How is time divided to calculate the kinetics functions?
The minimum is 1 bin/second and the maximum is a total of 512 bins. However, if you are computing certain statistics, FlowJo tries to adjust bin size to accommodate the stats. For example, if you are computing a percentile, it wants to have at least 10 events in each bin on the smaller side of the percentile. (For example, if you are computing median, FlowJo tries to have at least 20 events per bin; if you are doing the 10th or 90th percentile, it tries to have at least 100 events per bin, on average). For mean calculations, FlowJo wants to have at least 10 events per bin. If you are doing statistics on the events above threshold, these event numbers are increased by a factor of 3. Once FlowJo knows the minimum average number of events per bin necessary for the statistics, it computes the width of the bin accordingly. For example, if it wants 100 events per bin, and there are 10,000 events, then it will try to make 100 bins, as long as the absolute criteria (1 bin/second, max of 512 bins) are met. The reason for all of this is that we want to ensure that the computed statistics are valid. There is no validity to computing a 90th percentile (for example), if there are only 5 events in the bin.

4.3 Derived Parameters
4.3.1 What is the formula for Gain and Offset?
The formula for the final derived parameter is: D = offset + gain * P
where P is the current parameter, and offset and gain are what you specify.

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4.4 Cell Cycle
4.4.1 How do I remove doublets and cell fragments from my analysis?
We currently rely on you to manually gate out fragments and doublets from your samples.
4.4.2 Why doesn't G1, S and G2 add up to 100%?
The modeling algorithms contain some approximations, and are rounded numbers, so they won't add up to exactly 100% of the cells in the sample. Generally the sum of <G1,G1, S, G2, >G2 will be within one to two percent of 100%.
4.4.3 How does FlowJo choose the gates to create the G1, S and G2 subpopulations?
We use published algorithms, accepted in the flow community as the best methods available. The Watson model fits Gaussian curves to G1 and G2 and does not fit the S phase (Watson, Chambers, & Smith: A Pragmatic Approach to the Analysis of DNA Histograms with a Definable G1 Peak. Cytometry 8:1-8 (1987)). The Dean-Jett-Fox model fits Gaussian curves to G1 and G2 and a polynomial to S phase (Fox: A Model for the Computer Analysis of Synchronous DNA Distributions Obtained by Flow Cytometry. Cytometry 1:71-80 (1980)). The two population modeling is done using the Dean-Jett-Fox algorithm.

4.5 Proliferation
4.5.1 Why don't the numbers from the model and gates match?
The proliferation modeler constructs a series of Gaussian curves, the sum of which equals the distribution found in the data file. In the model, the tails of the curves will overlap, and those are added with the other curves. When gates are produced, hard boundaries are imposed at the points where the tails intersect, and the events are distributed into the population whose peak is closest to their position. This is not the same as the way the theoretical model is overlapping, and hence the totals will be different.
4.5.2 What do the division index, proliferation index and percent divided mean?
*Division Index is the average number of divisions that a cell (that was present in the starting population) has undergone. For example, if half of the cells in the starting population divided and the average number of divisions was 4, the Division Index would be 2.
*Proliferation Index is the average number of divisions that those cells which divided underwent. For example, if the average number of divisions for all the cells was 4, the Division Index would be 4.
*%Divided is the percentage of the cells of the original sample which divided (assuming that no cells died during the culture). For example, if half of the cells in the starting population have divided, the %Divided = 50%.
4.5.3 What does the RMS tell me about the model fit?
The Root Mean Square is the calculation of the accumulated error between the actual data and what the model would predict. The smaller the number for RMS, the better the fit. There are is no prescribed level of acceptable error for any type of experiment. You can only use the number as a relative indicator of how one model fits one sample vs. another.

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4.6 Population Comparison
4.6.1 What do the different statistics tell me for univariate comparison?
Several algorithms can be used to compare FACS data. The Kolmogorov-Smirnov (K-S) algorithm is a commonly used method to determine the confidence interval with which one can make the assertion that two flow cytometric univariate histograms are different.
The Overton cumulative histogram subtraction algorithm essentially subtracts histograms on a channel-by-channel basis to provide a percent of positive cells.
The Super-enhanced Dmax Subtraction (SED) is a new sophisticated algorithm by Bruce Bagwell to compute %Positives when comparing histograms.
A new comparison algorithm was recently developed for the comparison of distributions, called Probability Binning (PB). The PB comparison is related to the Cox chi-square approach, but with modified binning such that it minimizes the maximal expected variance. This algorithm has been shown to detect small differences between two populations and it does so in a quantitative way. For more information, see the pages on Population Comparison.
4.6.2 How do I determine the biologically meaningful T(X)?
T(X) is a statistic which provides an indication of the probability with which two distributions are different and also provides a metric by which multiple distributions can be ranked. The higher the value of T(X), the less like the control sample. The number represents how many standard deviations away from the control the test sample is, so a value greater than 4 would represent 99% confidence in a difference. However, the minimum value of T(X) that has biological significance depends on the nature of the data being analyzed and therefore needs to be determined empirically.
In order to determine a biologically meaningful T(X), you need to collect samples that you expect to be the same (e.g., the same sample collected twice, the same cell preparation divided and stained in duplicate, or cell preparations from animals of the same strain). Each of these sample pairs measures the variation that can occur between experimental samples. By comparing these pairs, you can determine a biologically meaningful cutoff value of T(X) to use in comparing "real" samples.
4.6.3 What is the appropriate number of events to collect for multiparameter population comparison?
The more events, the better will be your ability to discriminate different distributions. It doesn't matter how many events you collect; the statistic is computed based on the number of events. In other words, if you collect fewer events, then you will have a much lower T(x) for two distributions than if you had collected more events--indicating that the probability that these two distributions are different is not as high.
4.6.4 What number of bins should be used?
We suggest that the number of bin be roughly 10% of the number of channels in which the data is collected. By that rule, 8 bit data should be tested with 25 bins, and 10 bit data with 100. Be aware that 16 or even 32 bit data probably does not use the full range of data values available, and this rule does not apply to derive more that a few hundred bins.?

4.7 Calibration

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