IGC

BD FACSCalibur | Reservations

Optical configuration

  • FACSCalibur is equipped with a typical low-power aircooled 15mW blue (488nm) argon laser, from which 3 colors can be detected (FL1, FL2, and FL3), and a red (635nm) diode laser, which can detect APC or Cy5 (FL4). Therefore, 4 different colors can be detected simultaneously, based on the 2 excitation wavelengths and the wavelength range of each detector, shown in the figure below. Note that a band-pass filter designated as 530/30 indicates that it will detect light in the a 30nm range centered at 530nm, i.e., 515-545nm. LP stands for Long Pass filter, and indicates that it will detect light above the mentioned wavelength. A list of typical fluorescent labels detected in each channel is also displayed on the right.

Operating Procedures

  • Before Acquisition

    1. If machine is turned OFF, follow the Startup FACSCalibur procedure first (Blue Box on the right).
    2. Check if PBS tank is full. If necessary, refill tank.
      Clean up all spills. PBS is corrosive!
    3. Pressurize PBS reservoir (Push valve down [ON]).
    4. Check PBS filter for air bubbles. If necessary, vent filter by slightly tapping while opening and closing bleed valve.
    5. Start CellQuest and connect to cytometer.
      Acquire Connect to Cytometer (+B).
    6. If machine was disconnected, remove H2O tube (left by last user) and press PRIME (with the support arm to the right).
    7. Load your instrument settings, or set threshold to 52 in FSC (Cytometer Threshold, +2).
    8. Setup your plots (Instructions available in the UIC wiki-page).
    9. Open Status window (Cytometer Status, +4) and check if status is STANDBY, sheath fluid is OK, and sample voltage is approx. 10.23.
      If not, make sure PBS tank is well tightened, or check troubleshooting instructions in the UIC wiki-page .
    10. When status switches to STANDBY, switch to RUN and run PBS for about 1min in HIGH to check if machine is dirty.
      Though events may appear, it may not be dirt but air bubbles being released.
      Still, these are events that may or may not interfere with acquisition.
      Evaluate noisy events based on the location where they fall in your plots and on the rate at which you will be acquiring your sample.
    11. If too many events appear, it is probably due to the machine being dirty. Follow Cleaning Procedures in
      the After Acquisition section, and then repeat the previous step.
    12. You are ready to start Acquisition!
  • Startup FACScan


  • After Acquisition

    1. Run Cleaning Procedures (Blue Box on the right).
    2. Load your instrument settings, or set threshold to 52 in FSC.
      (Cytometer Threshold , +2).
    3. Remove H2O tube and run PBS for about 1min in HIGH to check if machine is dirty.
      Though events may appear, it may not be dirt but air bubbles being released.
      Still, these are events that may interfere with acquisition.
      Evaluate noisy events based on the location where they fall in your plots
      and on the rate at which your sample was being acquired.
    4. If too many events still appear follow Cleaning Procedures again, or call assistance.
    5. Switch to STANDBY and remove pressure (pull valve up [OFF]).
    6. REFILL PBS tank and replace PBS tube with H2O tube.
    7. Quit your acquisition files and REMOVE all your data.
      You may leave your acquisition documents (not the saved samples) for future use.
      However, the hard-disk is subject to routine cleaning, and files may be deleted without warning. The computer is NOT for data storage.
      Suggestion: You may remove your files while running Cleaning Procedures (s
      ee instructions on how to transfer files to the xservers).
    8. When finished, check if there is anybody reserved after you in the next 3 hours.
      IF YES : Leave everything running.
      IF NO : Quit CellQuest and shutdown the computer, and switch OFF FACSCalibur (flowcytometry button on right side).
      Remove your gloves, tubes and other belongings and clean up any spills or trash.
    9. If you have any doubts or need assistance call the facility's staff (ext. 251).
  • Startup FACScan