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University of Colorado Cancer Center

University of Colorado Cancer Center, A National Cancer Institute-designated Comprehensive Cancer Center
 

Facility Rules & Protocols

Facility Rules

  1. The Xenogen system is always on. Please do not turn off the Xenogen system computer or IVIS system components.
    • Please log off from the computer after each use to prevent your group from being overcharged for your time
    • Be sure to fill out the log binder to accurately record your user time, as well as logging on and off the system.
  2. You must supply your own luciferin.
    • You can purchase it for about $800/gram from Caliper Life Sciences.
    • If you expect to use only a small amount of luciferin, you can purchase 1mL aliquots of solution from Barbara Frederick, for $25/mL of solution. Please arrange with Barbara ahead of time so the luciferin solution will be available for you to purchase.
  3. You must keep the room clean and free of any animal hair, bedding, feces, fluids, etc., so please clean up when your experiment is done.
  4. We store a Xenogen system manual in the room for your convenience. If you remove the documents for copying, please return them to the room when you're done— within a few hours at most.
  5. Please acknowledge the facility in any publications arising from its use: In Vivo Gene Imaging Facility, University of Colorado Anschutz Medical Campus. Be sure to submit publications in which the facility is acknowledged so we can add them to annual reports justifying facility funding.
  6. Address problems, questions, concerns, comments or items you would like to see added to the facility to help you with your experiments to Barbara Frederick.

Data Processing

  • Please use the User Data folder on the desktop for short-tern data storage. Store your data in another folder inside the User Data folder named with the user's or PI's FIRST and LAST name.
  • Please remove data from the computer for long-term archiving and safekeeping after each imaging session. The Xenogen System computer has USB ports and CD-RW drive. Each image folder is about 4MB, so a USB device should be sufficient for most users.
  • We periodically delete data in the User Data folder with little advanced warning to ensure enough space for all users. You must take responsibility for your own data. We assume no responsibility for lost or corrupted data files.
    •

    Recordkeeping:

  • Fill out the IVIS system logbook when finished with your experiments. We use recorded usage hours as a gauge for performing routine maintenance to ensure the system functions properly. If you repeatedly fail to fill out the log, we will cancel your user privileges.

    • Luciferin Protocols

    The Xeno Corporation suggests the following protocols for preparing and administering luciferin. You may also use your own protocol.

    Preparing Luciferin for In Vitro Bioluminescent Assays

    Materials

    • D-Luciferin Firefly, potassium salt, 1.0 g/vial (Xenogen Catalog # XR-1001)
    • Sterile water
    • Complete media

    Procedure

    1. Prepared a 200X luciferin stock solution )30 mg/ml) in sterile water. Mix gently by inversion until luciferin is completely dissolved. Use immediately, or aliquot and freeze at -20°C for future use. You can either reconstitute the entire 1.0 g of D-Luciferin in 33.3 ml of sterile water to make the 30 mg/ml (200x) stock solution, or reconstitute the quantity of D-Luciferin necessary for an individual experiment.
    2. Prepare a 150 μg/ml working solution of D-Luciferin in pre-warmed tissue culture medium. Quick thaw 200X stock solution of luciferin and dilute 1:200 in complete media (150 ug/ml final).
    3. Aspirate media from cultured cells.
    4. Add 1x luciferin solution to cells just prior to imaging.

    Note: Incubating the cells for a short time at 37 C before imaging can increase the signal.

    Preparing Luciferin for In Vivo Bioluminescent Assays

    Materials

    • D-Luciferin, Firefly, potassium salt, 1.0 g/vial (Xenogen Catalog #XR-1001)
    • DPBS, w/o Mg2+ and Ca2+
    • Syringe filter, 0.2 um

    Procedure

    1. Prepare a fresh stock solution of luciferin at 15 mg/ml in DPBS. Filter sterilize through 0.2 um filter.
    2. Inject 10 μl/g of body weight intra-peritoneally. Each mouse should receive 150 mg luciferin/kg body weight. (e.g. For a 20 g mouse, inject 200 μl to deliver 1.5 mg of luciferin.). Inject the luciferin intra-peritoneally (i.p.) 10-15 minutes before imaging.
    3. Perform a luciferin kinetic study for each animal model to determine peak signal time.
    4. Anesthetize luciferin-injected mice with the attached XG-8 isofluorane device following standard procedures. The mice will be first placed into an induction chamber and anesthetized with a high initial dose and then maintained with a lower dose through a manifold located on the imaging stage. Mouse imaging can be conducted with the mice being continuously anesthetized.

    Instrument Disinfection

    The In Vivo Optical Gene Imaging Facility is a service with different users bringing in animals for experimentation. It is expected that all animals entering the facility will be cleared by veterinarians as being disease-free. However, as a precaution, the following steps should be performed to insure a clean and disease-free facility:

    1. Place absorbent paper available in the facility (the “blue diaper paper”) on the counters where animal work will be done. Perform all animal preparation on covered countertops.
    2. Place all animals on black paper on the stage in the Xenogen system (black paper is available in the facility in the drawer marked “Xenogen Supplies”).
    3. Dispose of all sharps in the sharps container.
    4. After using the facility, clean the counters (if necessary, clean the stage inside of the Xenogen IVIS system too). Use 70% isopropanol followed by Sporicidin.

    IMPORTANT: Only wipe the stage inside of the IVIS system with Kay-Dry wipes (this will prevent lint that can cause image artifacts from accumulating in the system). Kay-Dry wipes, 70% isopropanol, and Sporicidin spray are available in the facility.