CellQuest Pro Setup, Data Acquisition and Storage for the Consequent Runs of the Pre-set Experiment ("Return Users")
Note: If the same type of the experiment has been done on the same instrument before, the researcher could take advantage of the instrument's stability and experimental protocol consistency in the sample preparation. In this case the "Return User" should start with the Acquisition Template and Instrument Settings created in the previous experiment and make necessary adjustments based on the current controls.
Detector and Compensation Setup (for "Return Users")
- Double-click on the existing acquisition file (template) from the previously run experiment, which will automatically open the CellQuest Pro program.
- From "Acquire" menu > "Connect to Cytometer". The "Browser" and "Acquisition Control" windows appear automatically.
- Make sure the "Setup" mark on the "Acquisition Control" window is checked, so that the data will not be acquired (recorded).
- From "Cytometer" menu, open windows: "Detectors/Amps", "Compensation" and "Status".
- Load Instrument Settings from the previous experiment. Go to "Cytometer" > "Instrument Settings", click "Open", select the proper "xyC2 YYYYMMDD_InstrSettings" file or any FCS file from the proper experiment, click "Open". Then make sure to click "Set", then "Done".
- Check if the FSC Amp Gain and PMT voltageson all other detectors are still valid:
- Run an unstained cell control sample to make sure that all the populations appear in the original gates. Make minor adjustments to the detector values if necessary.
- Run single-color controls. Make sure that the positive cells from the single color controls remain on scale for each parameter. If the positive cells are "off scale" with this settings, adjust the corresponding PMT voltage down to get them on scale.
- Check if all the Compensations are still valid. Run single-color controls, one in a time and make minor adjustment in the "Compensation" window if necessary.
Note: For more details refer to the step 10 in the "CellQuest Pro Setup, Data Acquisition and Storage".
Data Acquisition (for "Return Users")
- In the "Acquisition Control" window, click "Pause" and "Abort". In order to record data uncheck the "Set up" mark.
- On the desktop create the new folder for today's experiment and name it uniformly "xyC2 YYYYMMDD" with your initials (xy) followed by C2 (for Calibur-2) and date – "YYYYMMDD".
- In the "Browser" window ("Windows" > "Browser"):
- Redefine the Directory to the today's folder "xyC2 YYYYMMDD" (just created on the desktop);
- Redefine the file namewith the same prefix:
- Custom Prefix: "xyC2 YYYYMMDD";
- File Count: "1".
- Redefine sample ID (for instance: unstained control).
- In the "Acquisition & Storage" window ("Acquire" > "Acquisition & Storage"):
- Check (and change if necessary) how many events and from which gate to count;
- Check (and change if necessary) from which gate the events to be stored;
- Check (and change if necessary) which parameters to store, Click "OK";
- Click "OK".
- Save this CellQuest Pro file as an updated Acquisition Template. Go to "File" > "Save Document As", name it as "xyC2 YYYYMMDD Acquisition", define the Directory to the today's folder "xyC2 YYYYMMDD", click "Save".
- Save updated "Instrument Settings". Go to "Cytometer" > "Instrument Settings", click "Save", name it as "xyC2 YYYYMMDD_InstrSettings", define the Directory to the today's folder "xyC2 YYYYMMDD".
- Acquire (record)the unstained and single-stained controls:
- Acquire the unstained control. When done, take it off and let the instrument to wash by keeping it on RUN without the tube;
- Change the sample ID from the "Browser" window, mix and put on the next sample and acquire. Make allsingle-color controls acquired in the similar manner.
- Proceed to the acquisition of the experimental samples data.
Note:User can retrieve the following widows at any time:
- "Palette", "Acquisition Control", "Inspector", and "Browser" (from "Windows");
- "Acquisition & Storage", "Parameter Description" and "Counters" (from "Acquire");
- "Detectors/Amps", "Threshold", "Compensation" and "Status" (from "Cytometer").
- After done with the experiment, make sure to re-save the Document file for the most updated template.
- Quit CellQuest Pro.