Bioluminescence Imaging of an Immunocompetent Mouse Model for Glioblastoma

Samenvatting

Source: Clark, A.J., et al. Bioluminescence Imaging of an Immunocompetent Animal Model for Glioblastoma. J. Vis. Exp. (2016)

This video demonstrates bioluminescence imaging to track glioblastoma progression in an immunocompetent mouse model, using luciferase-expressing tumor cells and luciferin to quantify tumor growth non-invasively.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Bioluminescence Imaging of GL261 Tumor Growth

1. Spray a generous amount of disinfectant (0.05% dimethyl ammonium chloride solution) on a clean paper towel. Use the disinfectant-soaked paper towel to thoroughly wipe the black sheet, nose cones, and divider inside the imaging chamber where the animals will be in contact with the device. Thoroughly wipe all surfaces with a clean, dry paper towel. Repeat this once more and wait 5 min.
2. Initialize the imaging station.
3. Anesthetize the mouse with a 300 μl mixture containing 200 μl of Ketamine/Xylazine and 100 μl of luciferin (D-Luciferin potassium salt, 150 mg/kg) by intraperitoneal injection.
4. Wait 10 min after injection and confirm if the mice are completely under anesthesia by pinching the tail. To set up the imaging system, select "Luminescent", "Auto" exposure time, and "Medium" binning. Place the mice on the imaging station and click the "Acquire" button to start the image. Ensure that the time after injection is consistent with each imaging session.
5. After the image is successfully acquired, an image window and tool palette should appear. Click the region of interest (ROI) or “ROI Tools” and select "auto" from the circle icon. To define ROIs, encircle the area of signal on the image and then take measurements of the ROI as a unit of photons/sec/sr/cm2.
6. Take the mice out of the imaging chamber and monitor them until they become ambulant and retain normal activity. Typically, recovery time is approximately 15 minutes. Do not return the mice to the cage with other animals until they fully recover from anesthesia.
7. Monitor tumor growth by bioluminescence imaging twice a week until animals present the following symptoms: weight loss (loss of more than 20% relative to the weight prior to implantation), inappetence (complete anorexia for 24 hr), and lack of sustained purposeful response to gentle stimuli (weak attempt to get up), at which time they should be euthanized.
8. Normalize luminescence values for each day against corresponding readings obtained on the first day of bioluminescence imaging (Figure 1A).
9. For statistical analysis, generate survival curves using the Kaplan-Meier estimator and compare the differences between survival curves using a log-rank test.

Resultaten

Figure 1. Luciferase expression does not affect in vivo tumor growth and animal survival. (A) Bioluminescence ...

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
D-Luciferin, Potassium salt	Gold Biotechonology	LUCK-100	Store away from light
Living Image software	Caliper Life Sciences	Contact for Quote	None
Xenogen Lumina	Caliper Life Sciences	Contact for Quote	None
Ketaset	Pfizer	NDC 0856-2013-01	Control substance
Xylazine	Sigma-Aldrich	23076-35-9	None
28 G needle (with syringe)	Fisher	22-004-270	AKA Insulin Syringe
Heating pad	Dunlap	HP950
PDI Alcohol prep pads	Fisher	23-501-711