Mice are injected by an intraperitoneal route with a Luciferin solution (15 mg/mL or 30 mg/kg, in PBS, dose of 150 mg/kg) that is allowed to distribute in awake animals for about 5-15 minutes. The mice are placed into a clear Plexiglas anesthesia box (2.5-3.5% isofluorane) that allows unimpeded visual monitoring of the animals; e.g. one can easily determine if the animals are breathing. The tube that supplies the anesthesia to the box is split so that the same concentration of anesthesia is plumbed to the anesthesia manifold located inside the imaging chamber. After the mice are fully anesthetized, they are transferred from the box to the nose cones attached to the manifold in the imaging chamber, the door is closed, and the “Acquire” button (part of Living Image program) on the computer screen is activated. The imaging time is between one to five minutes per side (dorsal/ventral), depending on the experiment. When the mice are turned from dorsal to ventral (or vice versa), they can be visibly observed for any signs of distress or changes in vitality. The mice are again imaged (maximum five minutes), and the procedure is complete. The mice are returned to their cages where they awake quickly.
Preparation and Injection of Luciferin for In Vivo Bioluminescent Assays
Materials
• D-Luciferin, Firefly, potassium salt, 1.0 g/vial, (Caliper Life Sciences Part Number #119222) DPBS, w/o Mg2+ and Ca2+
Note: One can either reconstitute the entire 1.0 g of D Luciferin in 66.6 mL of DPBS or reconstitute the quantity of needed for an individual experiment.
• Syringe filter, 0.2 μM
Procedure
1. Prepare a fresh stock solution of Luciferin at 15 mg/mL or 30 mg/kg in DPBS. Filter sterilize through a 0.2 μM filter.
2. Inject 10 μL/g of body weight. Each mouse should receive 150 mg Luciferin/kg body weight. See protocol below. (e.g. For a 10 g mouse, inject 100 μL of 15 mg/mL to deliver 1.5 mg of Luciferin).
3. Inject the Luciferin intra-peritoneally (i.p.) 5-15 minutes before imaging.
Note: A Luciferin kinetic study should be performed for each animal model to determine peak signal time after Luciferin administration.
Intraperitoneal (i.p.) Injection
of Luciferin
Preferred Site
Animal’s lower left abdominal quadrant.
Needle Size
25 gauge, usually used with 1 cc syringe
Volume
100 μL of Luciferin (15 mg/mL stock) per 10 grams of mouse body weight.
Note: 1 mL i.p. injection of a nonirritating solution is easily tolerated.
Position of Animal
Manually restrained, dorsal recumbency (abdomen side up), with cranial (head) end of animal pointed down.
Injection
Needle should be bevel-side up and slightly angled when entering the abdominal cavity. Penetrate just through abdominal wall (about 4-5 mm). The tip of the needle should just penetrate the abdominal wall of the animal’s left lower abdominal quadrant
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