143
tumors generated from cell lines expressing very low levels of
reporter, the amount of luciferin can be increased to 150 mg/kg
body weight (i.e., 3.5–4 mg/mouse with 20–25 g average
mouse weight).
10. One may need to empirically determine the optimal inhibitor
concentration for the best signal-to-noise bioluminescence
detection in vivo. We usually test two to three different concentrations of the drugs in mouse tumor xenograft model to
find the optimal concentration that gives highest fold change
over vehicle control without being toxic to animals.
11. We create separate region-of-interest (ROI) for each tumor
based on its size and shape. We also make sure that this ROI
does not overlap with the ROI of any other tumors. We copypaste the same ROI for each tumor for counting total photon
flux for all the time points. We may move the position of the
ROI so that it covers the tumor (because the position of the
same animal with the same tumor might be slightly different
between different time points) but do not change the overall
shape or size of any ROI. This removes the chances of including background photon counts emanating from mouse skin.
12. We generally use four to five mice in an experimental group for
bioluminescence data acquisition on the IVIS Spectrum system. Based on the number of animals in a group, we choose a
stage level (distance of the CCD camera and the subject) and
use the same stage level for the whole experiment. Changing
the distance between the CCD camera and the subject height
between different time points or reads would lead to different
photon counts which will be difficult to analyze.
13. We usually use medium sensitivity settings with 15–60 s data
collection time for each read. One should acquire photon flux
without any saturated pixels. All the saturated pixels (above 10
8
counts) show the same value, and therefore, cannot be accurately analyzed.
14. We typically use the maximum photon flux emitted from each
tumor separately for all the calculations. Each tumor may show
the maximum emittance at different periods of time; therefore,
we usually perform sequential reads with a delay between each
read which allows us to collect the maximum photon flux for
all the tumors.
15. We typically use the maximum photon count for each tumor
for each time for the analysis. If a tumor does not respond at any
time-point, we omit that reading from the analysis. We analyze
and plot combined values of all the tumors from one treatment
group. Photon counts from vehicle (DMSO) or sham treated
animals are set as onefold and the ratios of other treatments are
counted as fold change from vehicle/sham treatment.
Molecular Imaging of ATM Kinase Activity
tumors generated from cell lines expressing very low levels of
reporter, the amount of luciferin can be increased to 150 mg/kg
body weight (i.e., 3.5–4 mg/mouse with 20–25 g average
mouse weight).
10. One may need to empirically determine the optimal inhibitor
concentration for the best signal-to-noise bioluminescence
detection in vivo. We usually test two to three different concentrations of the drugs in mouse tumor xenograft model to
find the optimal concentration that gives highest fold change
over vehicle control without being toxic to animals.
11. We create separate region-of-interest (ROI) for each tumor
based on its size and shape. We also make sure that this ROI
does not overlap with the ROI of any other tumors. We copypaste the same ROI for each tumor for counting total photon
flux for all the time points. We may move the position of the
ROI so that it covers the tumor (because the position of the
same animal with the same tumor might be slightly different
between different time points) but do not change the overall
shape or size of any ROI. This removes the chances of including background photon counts emanating from mouse skin.
12. We generally use four to five mice in an experimental group for
bioluminescence data acquisition on the IVIS Spectrum system. Based on the number of animals in a group, we choose a
stage level (distance of the CCD camera and the subject) and
use the same stage level for the whole experiment. Changing
the distance between the CCD camera and the subject height
between different time points or reads would lead to different
photon counts which will be difficult to analyze.
13. We usually use medium sensitivity settings with 15–60 s data
collection time for each read. One should acquire photon flux
without any saturated pixels. All the saturated pixels (above 10
8
counts) show the same value, and therefore, cannot be accurately analyzed.
14. We typically use the maximum photon flux emitted from each
tumor separately for all the calculations. Each tumor may show
the maximum emittance at different periods of time; therefore,
we usually perform sequential reads with a delay between each
read which allows us to collect the maximum photon flux for
all the tumors.
15. We typically use the maximum photon count for each tumor
for each time for the analysis. If a tumor does not respond at any
time-point, we omit that reading from the analysis. We analyze
and plot combined values of all the tumors from one treatment
group. Photon counts from vehicle (DMSO) or sham treated
animals are set as onefold and the ratios of other treatments are
counted as fold change from vehicle/sham treatment.
Molecular Imaging of ATM Kinase Activity