Animal experiments were approved by the Animal Care and Use Committee at the University of Virginia and conformed to the National Institutes of Health guidelines for the use of animals in research. U87 glioma cells or U87 glioma cells that had been stably transfected with an mCherry reporter gene (i.e., U87mCherry) were implanted into 6- to 8-week-old male athymic nude mice purchased from Charles River Laboratories. B16F1ova melanoma cells were implanted in 8- to 10-week-old C57BL/6 mice, which were also purchased from Charles River Laboratories. Mice were anesthetized with a mixture of ketamine (40 mg/kg; Zoetis, Kalamazoo, MI) and Dexdomitor (0.2 mg/kg; Zoetis, Kalamazoo, MI) in 0.9% sterile saline and situated on a stereotaxic frame. Buprenorphine analgesic was subcutaneously administered. The surgical site was prepared with alternating scrubs of alcohol and iodine, and an incision was made at the midline of the scalp. A drill was used to create the burr hole located 2 mm to the right and 0.5 mm anterior to the bregma. A 10-μl Hamilton syringe with a 26-gauge needle was loaded with tumor cells (1.5 × 108 U87 cells/ml and 2.0 × 108 B16F1ova cells/ml). The needle tip was lowered to a depth of 4 mm below the skull surface and then withdrawn 1 mm to a final depth of 3 mm. A total volume of 2 μl of tumor cells (3 × 105 U87mCherry and U87 cells or 4 × 105 B16F1ova cells) were injected over 4 min. After one additional minute, the needle was slowly removed from the brain. The incision was closed with sutures and animals were given Antisedan to reverse the anesthesia.

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