142
immunoblotting using antibodies against ATM and its substrate
CHEK2.
3. We generally select for clones that exhibit low to moderate
bioluminescence after adding luciferin. Since it is an activatable
reporter that shows an increase in the light with inhibition of
the ATM kinase, clones expressing the reporter in very high
abundance may not yield high signal to background and thus
may show a limited fold activation in response to inhibitors.
4. For picking up the clones, complete growth media with antibiotic is aspirated from the tissue culture dishes containing singlecell clones. Sterilized filter paper discs pre-wetted in trypsin are
transferred to clones using sterilized forceps in a laminar flow
hood. After 4–5 min, the discs are lifted from the tissue culture
plate and swirled around in a 24-well plate containing complete
growth media with half the concentration of antibiotic used for
clone selection. Generally, 12–24 single-cell clones are picked.
Forceps are sterilized either by heat or by dipping them in
ethanol for 5–10 min. Make sure to let the ethanol evaporate
before using the forceps to pick up the clones.
5. For expanding and maintaining of stable cell lines, we use half
the concentration of G418 than that was used for selection.
The G418 concentration for stable clone selection should be
empirically decided.
6. The N-Luc and C-Luc fragments used in the construction of the
complementation-based ATM reporter are derived from a firefly
luciferase that has been optimized to work in mammalian cells
at the physiological temperature of 37 °C. Therefore, all the
bioluminescence acquisitions should be performed at 37 °C.
7. Since this reporter is based on the complementation of the
light-generating enzyme luciferase, it works only in live cells
under physiological conditions. Methods wherein cell lysate is
used to measure the bioluminescence signals are incompatible
with this reporter system and will not be able to yield any
detectable change in signal to background (under different
treatment conditions).
8. The Envision system is built with ultra-sensitive luminescence
detection technology; thus 0.01–0.1 s measurement for each
well is usually sufficient. The detection time can be increased if
the signal is very weak and desired signal-to-noise is not reached
with lower detection times. Furthermore, the bioluminescence
signal from cAMP-Glo reagent is stable for a prolonged period
of time, allowing us to measure the kinase activity of ATM for
longer periods.
9. In our hands, we find that 400 μg luciferin/mouse gives us the
best signal-to-background bioluminescence readings for split
firefly luciferase-based kinase reporters. For smaller tumors or
Shyam Nyati et al.
immunoblotting using antibodies against ATM and its substrate
CHEK2.
3. We generally select for clones that exhibit low to moderate
bioluminescence after adding luciferin. Since it is an activatable
reporter that shows an increase in the light with inhibition of
the ATM kinase, clones expressing the reporter in very high
abundance may not yield high signal to background and thus
may show a limited fold activation in response to inhibitors.
4. For picking up the clones, complete growth media with antibiotic is aspirated from the tissue culture dishes containing singlecell clones. Sterilized filter paper discs pre-wetted in trypsin are
transferred to clones using sterilized forceps in a laminar flow
hood. After 4–5 min, the discs are lifted from the tissue culture
plate and swirled around in a 24-well plate containing complete
growth media with half the concentration of antibiotic used for
clone selection. Generally, 12–24 single-cell clones are picked.
Forceps are sterilized either by heat or by dipping them in
ethanol for 5–10 min. Make sure to let the ethanol evaporate
before using the forceps to pick up the clones.
5. For expanding and maintaining of stable cell lines, we use half
the concentration of G418 than that was used for selection.
The G418 concentration for stable clone selection should be
empirically decided.
6. The N-Luc and C-Luc fragments used in the construction of the
complementation-based ATM reporter are derived from a firefly
luciferase that has been optimized to work in mammalian cells
at the physiological temperature of 37 °C. Therefore, all the
bioluminescence acquisitions should be performed at 37 °C.
7. Since this reporter is based on the complementation of the
light-generating enzyme luciferase, it works only in live cells
under physiological conditions. Methods wherein cell lysate is
used to measure the bioluminescence signals are incompatible
with this reporter system and will not be able to yield any
detectable change in signal to background (under different
treatment conditions).
8. The Envision system is built with ultra-sensitive luminescence
detection technology; thus 0.01–0.1 s measurement for each
well is usually sufficient. The detection time can be increased if
the signal is very weak and desired signal-to-noise is not reached
with lower detection times. Furthermore, the bioluminescence
signal from cAMP-Glo reagent is stable for a prolonged period
of time, allowing us to measure the kinase activity of ATM for
longer periods.
9. In our hands, we find that 400 μg luciferin/mouse gives us the
best signal-to-background bioluminescence readings for split
firefly luciferase-based kinase reporters. For smaller tumors or
Shyam Nyati et al.