SPADE 3 is a analysis and visualization tool for single-cell cytometry data. This article describes the steps needed to get it up and running on a local machine; it assumes you already have some FCS files ready for analysis.


Note: If you encounter any problems installing the software, please contact us, adding "Attn: Rebecca Faill" to the Subject line.


  1. Download SPADE3
  2. Unzip downloaded file
  3. Double-click on the un-zipped file to start the installation Note: By default, SPADE will be installed to "/Applications/SPADE3_2016_10_10_mac/", and Matlab Runtime will be installed to "/Applications/MATLAB/MATLAB_Runtime/v85/"


Note: Some of the below components may already be installed on your system. If so, the installers should report this and let you exit.

  1. Download Windows .NET Framework 4, and install it.
  2. Download Windows SDK 7.1, and install it. Choose all of the default settings during the installation. If the installer gives you an error about a "pre-release version of .NET being already installed", ignore that error and continue with the install.
  3. Download SPADE 3, and install it. During installation, check the box "Add a shortcut to the desktop" on the first screen. For all other options, leave the default settings.

Running SPADE


Spade should always be run from a terminal window, so that status and error messages are visible. Some operations take a long time to complete, and without the terminal open it would appear that the software is frozen when it is in fact running fine.

  1. Open a terminal window. (How to Open the Command Line)
  2. Type the following and press enter, the software will start.
/Applications/SPADE3_2016_10_10_mac/application/ /Applications/MATLAB/MATLAB_Runtime/v85/


Double-click the SPADE desktop icon to start. The program takes a minute or so to load, don't be alarmed if it's just a blank terminal window for a while. Although a interactive dialog with buttons will eventually open, the terminal window will continue to be used to report the status of operations that take a long time to complete.

Converting Files So They Can Be Read By SPADE

In our testing, SPADE can't read FCS files as exported by FlowJo, or as they come off of the lab's machines. For SPADE to read the files, they have to be pre-processed. A free pre-processor we've found that works well is

Using Immport Galaxy to convert FCS files

  1. Sign up for a free account here:
  2. Log in. You should see a screen like this: img
  3. Upload all of your data files, using the "Upload Files" tool.
    -- Once the files are uploaded, they should appear in
    your "History" section on the right img
  4. Convert the files, using the ["Automated gating, Transformation and Conversion of FCS to Text"](https://immportgalaxy
    .org/tool_runner?tool_id=fcs_gate_trans_convert) tool. Tool should be set up as in this image,
    Note that multiple files can be selected at the top to create a batch job.
    img These settings will result in two output files for each input file uploaded. One will be a
    flowtext file, the other will be a properly formatted FCS file.
  5. Download the converted FCS files for each of your original data files. Downloading is
    available in the "history" tab on the right. First you will need to open up details for the file
    in question by clicking on its name, the download button looks like a floppy disc as shown here:

Move the converted FCS files to a new, empty directory. SPADE prefers to have a separate directory for each collection of files it processes.

Using SPADE to produce visualizations

Note: More detailed descriptions of all of the SPADE options and settings are available here.

  1. Run SPADE from the terminal window as described above.
  2. Select the directory (using the "browse" button at the top) in which you put your converted
    FCS files.
  3. SPADE will pop up a dialog confirming the files in the directory. You can close this without
    making changes.
  4. Click the "View/update algorithm parameters" button. This will bring up an options dialog. A
    discussion of each of the settings in this dialog is available here.
  5. Choose some markers to use in the SPADE tree from the section in the upper left.
  6. Close the settings dialog and click the "Compute local densities..." button. While the
    calculation is running, you can watch its progress in the terminal window. It will take a fair
    amount of time to run.
  7. Click the "Pool selected files" button - status will again be available in the terminal window.
  8. Click the "Clustering..." button - status will again be available in the terminal window.
  9. Once all above calculations are complete, you may set up a visualization by clicking the "View
    resulting SPADE tree" button. Setting up various visualizations is described here (Search for
    "Meaning of the plots" to find the appropriate section.)

Files exported by SPADE will appear in an "exported_results" subfolder inside of the folder containing your converted data files.