Ivis 200 station

Fertilized White Leghorn Chicken eggs were prepared for cancer cell inoculation as previously published (8 ). Each egg was inoculated with 2 million cells per condition. 6 eggs were run per condition and repeated in three independent experiments.
For in vivo imaging, 100 µl 15 mg/ml luciferase substrate was pippetted directly on the tumor and immediately imaged. Images were collected on a Xenogen IVIS 200 station using Living Image 4.2 software from PerkinElmer (Waltham, MA), set to an exposure time of 1 second with F/stop set to ½ and medium binning. Analysis of the data was performed in Microsoft Excel. Significance was calculated using Student’s unpaired two-tailed t-test.
Tissues from the lower CAM were dissected, collected, flash-frozen. Frozen samples were pulverized, and genomic DNA extracted following the protocol described in the DNeasy Blood and Tissue kit from Qiagen (Germantown, MD). The invaded human cells in the chicken cell background were quantified by performing quantitative RT-PCR for human-specific Alu sequence (Table 4), as previously reported (32 (link)).
Hyaluronic acid density and UGDH expression were assessed in the tumor and surrounding environment of the collected upper CAM samples using immunohistochemistry.

100 µl of 5 × 10⁵ luciferase labeled cells were injected into the lateral tail vein of 6-week-old female SCID-Beige mice purchased from Charles River (Wilmington, MA). Each week following tail vein injection anesthetized mice were given retro-orbital injections of 100 µl of 10 mg/ml luciferase substrate (GoldBio, Cat # LUCK-1G) and imaged immediately. Images were collected on a Xenogen IVIS 200 station using Living Image 4.2 software (PerkinElmer), set to an exposure time of 1 second with F/stop set to ½ and medium binning.