Lesson 11: Performing an Instrument Calibration

Now that the instrument has been prepared, it is time to perform an instrument calibration. This calibration must be performed whenever the instrument has been restarted. Before performing the Calibration it might be useful to review the CellQuest Pro User’s Guide to familiarize yourself with the software.

Daily Calibration
The Daily Calibration is a simple procedure. However, it is necessary in order to set the timing intervals between the primary and secondary laser as well as to verify that the instrument is functioning properly and that the flow cell is not obstructed. A special calibration template and instrument settings file has been prepared the instrument can be calibrated in the same manner every day.

Step One – Open CellQuest Pro

Log in to the Macintosh (username and password available from flow core staff). The CellQuest Pro icon has been added to the OSX Dock (image above) for you convenience. Simply select the CellQuest Pro icon and the software will open.

Once opened, you will be presented with a blank CQ Pro Document. From the Acquire menu choose Connect to Cytometer (above) in order to initiate communication with the instrument. Next, close the blank document by clicking the red close window tool in the upper left corner of the document.

Step Two – Open the Calibration Protocol

From the File menu, select Open Document and navigate to Data 1 > Setup. Choose the “Sphero Setup” document and click Open. The Calibration template will open on the desktop. Next, from the Cytometer menu, select Instrument Settings. Choose the “Sphero Instrument Settings” file and click Set followed by Done. This will load the appropriate instrument settings for the calibration.

Step Three – Time Delay Calibration

Obtain a sample of Spherotech Calibration particles from the flow core personnel. Load these particles onto the Sample Injection Probe (SIP) and hit the Run and High buttons on the front instrument panel.

Ensure that the Setup button in the CellQuest Pro Browser is checked and click Acquire. From the Acquire menu select Counters and verify that the flow rate is above 400 events/sec. If it is lower, verify that the instrument is running in High (front panel button). If it is in HIgh and the flow rate is still too low, ask the flow core personnel for a new sample of beads.

Once the machine is running with a flow rate above 400 events/sec, move to the Cytometer menu and select Time Delay Calibration followed by Calibrate. The instrument will provide an error message if there is a problem, otherwise, it will just beep to signify that the instrument has successfully calibrated. If there are any problems, please request assistance from flow core personnel.

Remove the calibration particles from the SIP. Place the dH2O tube back on the SIP, and place hit the Standby button on the front panel.

The instrument is now calibrated and ready for your experiment.

Wrapping Up
In order to asses your understanding of the material thus far, please email the answers to the following exercise to the address below.

• Prepare a FACSCalibur Calibration Quick Sheet listing all necessary steps in order. After review with (and possible correction by) the flow core director, maintain the quick sheet as a handy reference when preparing the instrument.
• Review the CellQuest Pro User’s Guide and the following movie (quicktime) which shows the creation of a simple experimental document as well as the running and adjustment of a negative and positive (PE) control sample. After reviewing this material. Phone the main lab at x73216 to schedule your first “hands-on” training session. Welcome to the University of Michigan Flow Cytometry Community.