After 6 hours of breathing room air or hypoxic gas, mice were anesthetized using isofluorane mixed with 21% oxygen (1.5% v/v) administered through a nose cone. Once breathing was stable, mice were placed on a heating pad for the duration of the experiment. Trans-illumination images, RFP fluorescence (for 4T1-RFP cells) and a background image at 525 nm corresponding to pre-NBDG injection were recorded. Any background fluorescence at this wavelength was due to FAD. 100 µl of 2-NBDG (2.5 mg/ml) dissolved in sterile saline was injected through the tail vein. The 2-NBDG fluorescence was recorded for 75 minutes as follows: continuously for the first 8 minutes, every 30 seconds for the next 30 minutes and every 3 minutes for the final 35 minutes of imaging.
High resolution images of 2-NBDG uptake and constitutive tumor RFP fluorescence were recorded using a Zeiss upright confocal microscope. 2-NBDG fluorescence was excited at 488 nm and imaged from 510–550 nm. RFP fluorescence was recorded from 580–620 nm. All images were collected using a 10×objective (NA = 0.45). Field of view for the first set of images (baseline –40 minutes) was 850×850 µm. After 40 minutes, field of view was 297×297 µm.
High resolution images of 2-NBDG uptake and constitutive tumor RFP fluorescence were recorded using a Zeiss upright confocal microscope. 2-NBDG fluorescence was excited at 488 nm and imaged from 510–550 nm. RFP fluorescence was recorded from 580–620 nm. All images were collected using a 10×objective (NA = 0.45). Field of view for the first set of images (baseline –40 minutes) was 850×850 µm. After 40 minutes, field of view was 297×297 µm.
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