D luciferin monopotassium salt

All melanomas were tagged with stable luciferase expression, enabling the quantitation of metastatic disease burden by bioluminescence imaging. Five minutes before performing luminescence imaging, mice were injected intraperitoneally with 100 μl of PBS containing Dluciferin monopotassium salt (40 mg/ml) (Goldbio) and mice were anaesthetized with isoflurane 2 min before imaging. All mice were imaged using an IVIS Lumina S5 (Perkin Elmer) with Living Image software (Perkin Elmer). After completion of whole-body imaging, mice were euthanized and individual organs (including the heart, lung, liver, pancreas, spleen, and kidney) were surgically removed and imaged. The exposure time ranged from 15 to 30 seconds, depending on the maximum signal intensity, to avoid saturation of the luminescence signal. To measure the background luminescence, a negative control mouse not transplanted with melanoma cells was imaged. The bioluminescence signal (total photon flux) was quantified with 'region of interest' measurement tools in Living Image software. Metastatic disease burden was calculated as observed total photon flux from all organs in xenografted mice minus background total photon flux in negative control mice. Negative values were set to 1 for purposes of presentation and statistical analysis.