General Info
Obtaining FlowJo
Compatibility
Error Messages
Upgrading to Version 4
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
General
Info
1.1 How can I learn about FlowJo?
1.2 What features does FlowJo have that other
analysis programs do not?
1.3 Does Tree Star offer training?
1.4 Obtaining FlowJo
1.4.1 How do I evaluate FlowJo? Is there
a demo version I can try?
1.4.1.1 I'm
running FlowJo in demo mode and the provided demo data files won't open!
1.4.1.2 I downloaded
FlowJo and it did not Unstuff itself correctly!
1.4.2 Can I try out FlowJo with my own
data before I buy?
1.4.3 How much does it cost? How do I
get it cheaper than that?
1.4.4 How do I order and pay for FlowJo?
1.4.5 Which License protection method
should I choose?
1.4.5.1 What
the heck is a dongle!?
1.4.5.2 ADB
or USB dongle?
1.4.6 How come my USB dongle is not
recognized by FlowJo?
1.4.7 Do I need an installer or extension
for my USB dongle to work with system OS X?
1.4.8 Can I upgrade to the current version
of FlowJo? How?
1.4.9 I have FlowJo documents from version
X - will they still work if I upgrade to version Y?
1.5.0 I have an ADB dongle and have a
new computer with a USB port. Can I exchange the dongle?
1.5 Compatibility
1.5.1 What types of computers does FlowJo
run on?
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?
1.5.4 What about OS X compatibility?
What about Windows compatibility?
1.6 Error Messages
1.6.1 What should I do when
I experience a FlowJo Error message.
1.6.2 FlowJo crashes before
start up (on computers running less than OS 8.5).
1.6.3 Why FlowJo doesn't
find its dongle after I've run CellQuest?
1.6.4 Getting your USB dongle
to work with OS X/9.2
1.6.5 My workspace has become
corrupted after a crash, can I recover it?
1.7 Upgrading
to Version 4
1.7.1 Why should I upgrade
to Version 4? How much does it cost?
1.7.2 Do I have to upgrade
to Version 4?
1.7.3 What license protection
options are available with Version 4? What operating systems does it run
on?
1.7.4 How do I exchange
my version 3 dongle for the new Version 4 dongle?
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?
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?
3.8 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?
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 website 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.
[top]
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 Unstuff
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.
20 Winding Way
San Carlos, CA 94070
Phone: 800 366 6045
Intl: 650-591-2854
Fax: 650-508-9186
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.
Currently
there are two types of dongles, ADB (shown at left) and USB (right).
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?
Currently 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.
To check the versions history, 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.
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 version 4 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 carborn 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.
1.7.4 How do I exchange my version 3 dongle
for the new Version 4 dongle?
Order the upgrade (either via the web
or by contacting us) and we will send you intsructions for exchanging
the dongles.
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 preference to "Use sample's
filename instead of Keyword". Open the Preferences (under the FlowJo
menu) and check the box next to "Use sample's filename instead of
Keyword".
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.
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.
[top]
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 textbox.
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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 recomputations 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
recompute.
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.
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 layed 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" pulldown
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 multiuser
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 relaunch 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 autosave, 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 Autosaving.
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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.
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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.