In Vivo Bioluminescence Imaging of Tumor Growth and Viability

On days one, three and eight, following the inoculation of bioluminescent tumor cells, the animals underwent in vivo imaging using the IVIS Lumina III imaging system (PerkinElmer, Waltham, MA, USA). The transfected 4T1 tumor cells express firefly luciferase which produces bioluminescence following the addition of its substrate (luciferin). In consideration of the imaging, a 15 mg/ml solution of D−luciferin sodium salt (Gold Biotechnology, St. Louis, MO, USA) in DPBS was administered i.p. in a 150 mg/kg dose. Animals were anesthetized using ketamine-xylazine three minutes following the administration of D-luciferin, and were imaged ten minutes postinjection using the following settings: auto acquisition, F/stop = 1 and Binning = 4. Data were analyzed using the Living Image software (PerkinElmer). Regions of interest (ROI) were drawn around the luminescent tumor signal automatically using identical signal thresholds. Total radiance, a calibrated unit of the luminescence (total photon flux per second), was calculated in each ROI and used for further statistical analysis.