138
1. We typically do all of our cell-based and in vivo bioluminescence
assays using the reporter expressing stable cell lines. We carefully select cell lines that represent an appropriate cellular and
biological context for our studies (see Note 2). Cells are transfected with the reporter plasmids and allowed to grow under
the antibiotic selection media. We typically pick 12–24 singlecell clones using sterilized filter paper discs and choose the best
clones by measuring bioluminescence in response to specific
kinase inhibitors (see Notes 3 and 4).
2. The three best reporter expressing stable cell lines (clones)
are expanded and frozen at low passages for future use.
Cells are maintained in 10 cm dishes with complete growth
media containing serum and the appropriate amount of G418
(see Note 5).
3. Stable cell lines are plated overnight in black-walled or whitewalled, clear-bottom 96-well plates for live cell assays. Cell
density should be 2500–10,000 cells per well in 100 μL complete growth medium with serum (see Notes 6 and 7).
4. Cell culture media is removed and ATMR expressing cells are
treated with different concentrations of ATM inhibitors such
as caffeine, KU-60019, KU-55933, or CGK733 in serum-free
media (100 μL per well).
5. After 5 min, 2.5 μL d-luciferin (black-walled plates) or cAMPGlo reagent (white-walled plates) is added by multichannel
pipette into each well for a working concentration of 100 μg/mL
of firefly luciferase substrate.
6. Black-walled 96-well plates are imaged on the IVIS imaging
system as soon as possible after adding luciferin. Typically,
bioluminescence is acquired for 30–60 s at medium binning.
For a time-course, the images are acquired with 3–10 min
delay between the reads (Fig. 3a, b).
7. The white-walled plates are read on the Envision system right
after the addition of the substrate. Generally, each well of the
plate is read for 0.01–1.0 s. For a longer time-course activity
measurement, a delay of 15–60 min between each read is set
(Fig. 3c). For each read, the robot takes the plate out from the
incubator, loads it on the reader where the plate is read, and is
transferred back to the incubator until the next time point
(see Note 8).
8. Quantify bioluminescence acquired on IVIS system by regionof- interest (ROI) analysis using Living Image software. The
bioluminescence data from Envision system is automatically
saved in quantitative form in tab-delimited file format.
9. Since radiation activates ATM within minutes, the bioluminescence activity of ATMR can be evaluated within 15 min after
irradiation.
3.2 Cell-Based
Bioluminescence
Imaging of ATM
Kinase Activity
Shyam Nyati et al.
1. We typically do all of our cell-based and in vivo bioluminescence
assays using the reporter expressing stable cell lines. We carefully select cell lines that represent an appropriate cellular and
biological context for our studies (see Note 2). Cells are transfected with the reporter plasmids and allowed to grow under
the antibiotic selection media. We typically pick 12–24 singlecell clones using sterilized filter paper discs and choose the best
clones by measuring bioluminescence in response to specific
kinase inhibitors (see Notes 3 and 4).
2. The three best reporter expressing stable cell lines (clones)
are expanded and frozen at low passages for future use.
Cells are maintained in 10 cm dishes with complete growth
media containing serum and the appropriate amount of G418
(see Note 5).
3. Stable cell lines are plated overnight in black-walled or whitewalled, clear-bottom 96-well plates for live cell assays. Cell
density should be 2500–10,000 cells per well in 100 μL complete growth medium with serum (see Notes 6 and 7).
4. Cell culture media is removed and ATMR expressing cells are
treated with different concentrations of ATM inhibitors such
as caffeine, KU-60019, KU-55933, or CGK733 in serum-free
media (100 μL per well).
5. After 5 min, 2.5 μL d-luciferin (black-walled plates) or cAMPGlo reagent (white-walled plates) is added by multichannel
pipette into each well for a working concentration of 100 μg/mL
of firefly luciferase substrate.
6. Black-walled 96-well plates are imaged on the IVIS imaging
system as soon as possible after adding luciferin. Typically,
bioluminescence is acquired for 30–60 s at medium binning.
For a time-course, the images are acquired with 3–10 min
delay between the reads (Fig. 3a, b).
7. The white-walled plates are read on the Envision system right
after the addition of the substrate. Generally, each well of the
plate is read for 0.01–1.0 s. For a longer time-course activity
measurement, a delay of 15–60 min between each read is set
(Fig. 3c). For each read, the robot takes the plate out from the
incubator, loads it on the reader where the plate is read, and is
transferred back to the incubator until the next time point
(see Note 8).
8. Quantify bioluminescence acquired on IVIS system by regionof- interest (ROI) analysis using Living Image software. The
bioluminescence data from Envision system is automatically
saved in quantitative form in tab-delimited file format.
9. Since radiation activates ATM within minutes, the bioluminescence activity of ATMR can be evaluated within 15 min after
irradiation.
3.2 Cell-Based
Bioluminescence
Imaging of ATM
Kinase Activity
Shyam Nyati et al.