Lesson 10: Preparing the FACSCalibur for Operation

We are now switching our focus from basic flow cytometric principles to operation of the core’s FACSCalibur cell analyzer. This instrument is available to trained investigators, 24 hours a day/7 days a week. Appointments must by scheduled by calling the main lab at x73216. All appointments are prescheduled. No walk-ins are available!

The FACSCalibur

The FACSCalibur is located in the main lab (room 7416 of the Cancer and Geriatrics Center). Data acquisition is accomplished via the CellQuest Pro application which runs on Max OSX.

Looking Closer at the instrument itself, there are three areas to note: the fluid drawer, the sample chamber and the run controls.

Instrument Startup

Starting the FACSCalibur is a three-step process:

  1. Checking fluidic levels
  2. Electronic Startup
  3. Fluidic Prime

While a simple procedure, each involves a number of sub-steps. The complete procedure is explained in detail below.

Step One – Fluidic Levels

Before turning on the FACSCalibur, the sheath tank must be filled and the waste tank must be emptied.

Upon opening the fluid drawer, you will see the two tanks. The tank on the left (under the black plate) is the sheath tank. This contains the saline which carries the sample through the flow cell. The tank on the right is the waste tank. All fluid passing through the flow cell will end up in this tank.

Taking a closer look at the sheath tank, you should notice the black aluminum safety shield, the tank vent switch, and the fluid probe. If the vent switch is facing forward, the sheath tank is pressurized. Do not remove the safety shield or open the tank while pressurized! To depressurize the tank, simply flip the vent switch so that it is facing rearward into the instrument.

Once depressurized, unscrew and remove the fluid probe from the tank. As it is still connected to the fluidic drawer by one electrical and two fluidic tubes, simply move it to the side out of your way. Next, remove the black safety plate by pushing the plate down and back. When handling this plate, be aware that while the edges are rounded, it is still possible to scrape and/or cut yourself if it is handled in a careless manner. At this point, the tank can be removed for filling.

Fill the tank from the blue bottles near the sink labeled SHEATH. If these bottles are empty, speak to a flow core technician regarding access to the bulk saline supplies. As the sheath tank is pressurized during operation, be sure to leave sufficient air volume within the tank to allow for stable pressurization. Use the image above as a filling guideline.

Once the tank is full, return it to the instrument. Afterwards, complete the following in the order listed:

  1. Replace the black safety shield.
  2. Replace the fluid probe. Ensure that the cap is secure.
  3. Toggle vent swtich to pressurize the tank. The swtich should be facing forward.

Whenever the sheath tank is filled, the waste tank must be emptied. The waste tank is not pressurized and does not contain a safety shield. As such, simply remove the fluid probe and set aside. Remove the tank from the fluid drawer and dispose of waste fluid in the sink. Rinse the tank with tap water. Afterwards, add 50ml of bleach to the tank (Bleach is located under the sink), and return the tank to the fluid drawer. Replace the fluid probe and close the drawer.

Step Two – Electronic Startup

In order to perform the electronic start-up, turn on the FACSCalibur unit. The switch is located in the lower, back corner of the right-hand instrument panel. The switch will light up when in the “On” position. After turning the instrument on, reboot the Macintosh computer (Username and password are available from flow core personnel).

Step Three – Fluidic Prime

The final preparation step is the fluidic prime which consists of the following steps:

  1. Remove dH2O tube form Sample Injection Probe (SIP)(1. Grasp tube with fingers 2. Push swing arm to the left (see image above) 3. Pull tube downwards off of SIP. Be sure no tto hit or bend the SIP tubing.)
  2. Press PRIME button on front panel. The button will turn red during the prime cycle and bubbles will be seen on the tip of the SIP as the flow cell is flushed. The red light will go out once the cycle is completed.
  3. Repeat prime cycle two more times.
  4. Replace dH20 tube on SIP, and hit the STANDBY button on the front panel.

At this point the FACSCalibur is prepared and ready for daily calibration.

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. After receiving this I will provide you with access to the next module.

  • Prepare a FACSCalibur Preparation 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.
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