140
PBS; 400 μg per mouse) anesthetized with 1–2% isoflurane for
5 min (see Note 9).
3. Transfer mice to the bioluminescence instrument, where they
are maintained under anesthesia, and acquire bioluminescence.
We typically acquire data on five mice at once isolated by a
plastic separator. Generally, a 15–30 s acquisition at medium
sensitivity is sufficient. We typically acquire data for 10–20
reads with a 1–5 min delay between the reads to cover the bioluminescence peak from all the tumors in each of the mice.
4. Treat the mice with appropriate inhibitors such as KU-55933
(both 25 mg/kg), or activators such as radiation (5 Gy) and
monitor bioluminescence over time. Vehicle control (DMSO)
or sham-irradiated mice should be used as control (Figs. 3c
and 4b; see Note 10).
Fig. 4 In vivo measurement of ATM kinase activity in mouse tumor xenograft model. (a) CD-1 nude mice harboring
D54-ATMR WT tumor xenografts were injected with luciferin and bioluminescence was acquired as described
3 h before treatments. (b) The animals were injected with KU-55933 (25 mg/kg) or vehicle control (DMSO) and
bioluminescence was acquired 1, 4, 8, and 24 h posttreatment. The ATM reporter fold activation upon ATM
inhibition is plotted over mock treatment. (c) Similarly, mouse harboring D54-ATMR WT tumor xenografts
were whole body irradiated with 5Gy of radiation or sham irradiated and bioluminescence was measured for
up to 24 h. About 70% decrease in the reporter activity was observed 8 h post irradiation
Shyam Nyati et al.
PBS; 400 μg per mouse) anesthetized with 1–2% isoflurane for
5 min (see Note 9).
3. Transfer mice to the bioluminescence instrument, where they
are maintained under anesthesia, and acquire bioluminescence.
We typically acquire data on five mice at once isolated by a
plastic separator. Generally, a 15–30 s acquisition at medium
sensitivity is sufficient. We typically acquire data for 10–20
reads with a 1–5 min delay between the reads to cover the bioluminescence peak from all the tumors in each of the mice.
4. Treat the mice with appropriate inhibitors such as KU-55933
(both 25 mg/kg), or activators such as radiation (5 Gy) and
monitor bioluminescence over time. Vehicle control (DMSO)
or sham-irradiated mice should be used as control (Figs. 3c
and 4b; see Note 10).
Fig. 4 In vivo measurement of ATM kinase activity in mouse tumor xenograft model. (a) CD-1 nude mice harboring
D54-ATMR WT tumor xenografts were injected with luciferin and bioluminescence was acquired as described
3 h before treatments. (b) The animals were injected with KU-55933 (25 mg/kg) or vehicle control (DMSO) and
bioluminescence was acquired 1, 4, 8, and 24 h posttreatment. The ATM reporter fold activation upon ATM
inhibition is plotted over mock treatment. (c) Similarly, mouse harboring D54-ATMR WT tumor xenografts
were whole body irradiated with 5Gy of radiation or sham irradiated and bioluminescence was measured for
up to 24 h. About 70% decrease in the reporter activity was observed 8 h post irradiation
Shyam Nyati et al.