IGC

Analysis Software

In this section we provide simple reviews and help on different commercial and free softwares for analysis of flow cytometry data.

    • CellQuest
    • Cell Quest

      Tips for acquiring and analyzing with CellQuest.

    • FlowJo
    • FlowJo

      Reviews on how to handle keywords, concatenate files, compensation tutorials, etc. Information will be added regularly.

    • Summit
    • Summit

      Tips on how to handle acquisition and analysis, from database handling to compensations. Information will be added regularly.

    • FACSDiva
    • FacsDiva

      Tips on how to handle acquisition and analysis, from file managing to compensations.

      Coming soon!

Acquisition with CellQuest Interface

Load CellQuest

  • You can load CellQuest in two ways. Either by opening a previously saved acquisition page file or by clicking directly on the CellQuest icon in the Desktop. In this latter case, a new document page should appear. If not, you can go through File → new in CellQuest menu.

    To perform acquisition, you must first create plots that will allow you to visualize your cells, set regions and gates. Choose the plot type from the Plots menu. Also, for quick access you can activate the plots/tools poup-up (palette, left in the figure), if not seen on the screen: windows→show palette.


Connect to Cytometer

  • acquisition_control_bar_setup.jpg
  • First of all, you need to connect the computer to the cytometer. Acquire → connect to cytometer. If the connect to cytometer function is not available, then shutdown both the computer and the cytometer and perform again the turning on procedure in the right order (cytometer→computer). Once the connection is established the Acquisition Control Dialog Box with acquisition control functions (Setup, Acquire, Restart, Save, Abort) will appears. The setup box should have an X mark until you are ready to start saving data. Move the window to the side.

If this box do not appears by default, then go through windows→show acquisition control bar.

The counter window is also under Acquire.

counter_small.jpg counter_big.jpg


Retrieve Instrument Settings

CytometerInstrument SettingsOPEN → double click on a file → SET (wait for 5 seconds, while the world turns) → DONE.

cytometer_options.jpg instrument_settings.jpg


To Check and Adjust Sample Settings

The Setup box should have an X, while you run your negative background etc. to adjust the high voltages and compensations. To make the adjustments in CellQuest, select CytometerDetectors/Amps. Select Threshold and Compensation, if these will be adjusted. Activate the four colour option (lower left corner of Detectors/Amps) if you intend to detect fluorescence in FL4 (this turns on the red laser). The Status bar is also found in Cytometer

cytometer_options.jpg

detector_amps_scalibur.jpgcompensation_scalibur.jpgthreshold_scalibur.jpg status_bar.jpg


To Save Data Files

Assing Data Filename and Count

AcquireParameter DescriptionFolder(FACSData/user folder)→ File (custom prefix = initials+YYMMDD, and the file count = 1) → Enter sample information in the comment box. Enter sample information in the Sample ID box, if you will be using CellQuest for analysis. Information can be entered in both places.

picture3.jpgparameter_description.jpg file_name_editor.jpg

Assign Storage Information

AcquireAcquisition and StorageAcquisition Gate (Accept all), Collection criteria (Event count = # of cells to be counted, total or in a gate -acquisition will stop when this number is reached). Storage gate (All or gated) If a gate is selected, events outside the gate will NOT be saved for future analysis. Resolution (256 or 1024) → Parameters saved (turn off the parameters not in use) → OK.

picture2.jpg


Run Samples

In the cytometer, turn the knob from Standby to Run. Swing the arm to the side. Remove sheath tube. Put on your sample tube. Swing the arm to the left. Pulse lights should appear if cells are flowing (only in FACScan). Select Acquire in the acquisition control box. Make adjustments as necessary. When putting the next tube on, there is no need to switch to Standby. The sample probe is continually flushing while in the run mode. When satisfied with the settings, they must be saved, if you want to use them again.


To Save New Instrument Settings

CytometerInstrument SettingsSAVE → Find settings folder within user screens, settings and data folder → type in appropriate name → SAVEDONE.


To Acquire Data

Acquisition Control Box → PauseAbort → remove the X from the Setup box. When the correct sample is flowing, select Acquire. A tone will sound when collection is finished and the file count will increment. Change the comment or sample ID info and run the next sample.

acquisition_control_bar_steup.jpg acquisiton_bar_acquire.jpg


To Quit CellQuest

FileQuitSAVE if you wish to keep screen changes made during this run. The settings are NOT saved as part of the screen. They must be saved separately as explained above.

FlowJo Resources

FlowJo has many resources available. I find that usually the fastest way to find the solution is to google my question using the keyword "FlowJo". Almost always, there is already an answer, typically in FlowJo's amazing Blog: The Daily Dongle.

Still, you may find more help in the following sites:

  1. FlowJo Website: Check the online manuals, tutorials, and other help in this website.
  2. The Daily Dongle: An incredibly updated and comprehensive blog, full of tips, tricks, and howto's on FlowJo usage by Maciej Simm from Treestar, Inc.
  3. FlowJo Discussion Group at European Cytometry Network (ECN): This is a general discussion group for FlowJo users, accessible only if you are registered at the ECN. To request an invitation to register send an email to the facility's staff.

Analysis: Changing parameter names after acquisition

To change the name of your parameters after acquisition, you should do the following steps:

    1. Load your data file by double-clicking on its name in the Database Samples Window.
    2. Right-click on the data filename in the same Database Samples window and select "Properties...".
    3. In the Sample Properties window, select the Parameter tab.
    4. Select the parameter you want to change using the drop-down menu.
    5. Change the "Long Name" field to the desired name.
    6. Repeat steps 4-5 for all the other parameters you want to change.
    7. Click OK. You should see the new names in the list of parameters by right-clicking the axis of your histograms.
  • Changing Parameter Names in Summit

Coming Soon.