Tips for acquiring and analyzing with CellQuest.
In this section we provide simple reviews and help on different commercial and free softwares for analysis of flow cytometry data.
Tips on how to handle acquisition and analysis, from database handling to compensations. Information will be added regularly.
Tips on how to handle acquisition and analysis, from file managing to compensations.
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.
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.
Cytometer → Instrument Settings → OPEN → double click on a file → SET (wait for 5 seconds, while the world turns) → DONE.
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 Cytometer → Detectors/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
Acquire → Parameter Description → Folder(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.
Acquire → Acquisition and Storage → Acquisition 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.
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.
Cytometer → Instrument Settings → SAVE → Find settings folder within user screens, settings and data folder → type in appropriate name → SAVE → DONE.
Acquisition Control Box → Pause → Abort → 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.
File → Quit → SAVE 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 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:
To change the name of your parameters after acquisition, you should do the following steps: