265
7. Day 5: The transduced cells should have reached a confluence
of ~80% before continuing. Switch off ambient light and turn
on the safe lights. Aspirate the medium and wash the cells once
with PBS before adding 150 μl trypsin-EDTA solution. Place
cells back into incubator for ~3 min or until they detach.
8. Wash cells off the plate with 2 ml culture medium, transfer
them to a new 10-cm plate, and add culture medium up to a
total of 10 ml (see Note 11). Keep cells in incubator until they
have reached ~80% density.
9. The cells now stably express the transduced constructs (see
Note 12). Seed the cells (10
5
and 0.3 × 10
5
cells/cm
2
for
MCF7 and C2C12 cells, respectively) in either 6-well plates
(for Western blot analysis) or on coverslips in 24-well plates
(for fluorescence microscopy). Again, include a replicate on a
separate plate for each condition as dark control.
1. Day 1: Start with optoAKT expressing cells on 6-well or
24-well plate transfected/seeded the day before (see
Subheadings 3.1.1 and 3.1.2, respectively), which should have
a confluence of ~80% in the evening. Replace culture medium
with starvation medium (see Note 13) and place plates back
into incubator. It is important that the cells are not exposed to
any source of irradiation (390–480 nm) that would inadvertently excite the optogenetic system during starvation.
2. Day 2: In the morning (~12–15 h post starvation) cells are
ready for the experiment. The exact conditions of irradiation
vary depending on the desired readout.
1. For analysis of optoAKT by Western blotting set the intensity
of the LEDs in the light box to 150 μmol/(m
2
s) and irradiate
the cells 15 times for 5 s/min or constantly with a lower intensity (see Note 14). Intensities higher than 50 μmol/(m
2
s) may
induce photo-cytotoxic effects when applied for extended time
periods. Before further processing the cells ensure that ambient light is turned off and safe light is turned on (see Note 15)
and that there are no additional sources of light which accidentally could activate the optogenetic system.
2. Aspirate medium and wash cells once with PBS before adding
250 μl ice-cold modified TENT cell lysis buffer. Gently shake
the plates to evenly distribute the lysis buffer. Incubate the
plates for 10 min on ice.
3. Use a cell scraper to detach the cells and transfer the cell lysate
into a precooled 1.5 ml Eppendorf tube. Sonicate the lysate for
15 min (30 s/min pulses) at 4 °C to fragment DNA and lower
viscosity. In the next step, centrifuge lysates for 15 min at
10,000 × g and 4 °C to pellet the insoluble components.
3.2 Light Experiment
and Readout
3.2.1 Western Blot
Analysis
Optogenetic Control of Protein Kinases
7. Day 5: The transduced cells should have reached a confluence
of ~80% before continuing. Switch off ambient light and turn
on the safe lights. Aspirate the medium and wash the cells once
with PBS before adding 150 μl trypsin-EDTA solution. Place
cells back into incubator for ~3 min or until they detach.
8. Wash cells off the plate with 2 ml culture medium, transfer
them to a new 10-cm plate, and add culture medium up to a
total of 10 ml (see Note 11). Keep cells in incubator until they
have reached ~80% density.
9. The cells now stably express the transduced constructs (see
Note 12). Seed the cells (10
5
and 0.3 × 10
5
cells/cm
2
for
MCF7 and C2C12 cells, respectively) in either 6-well plates
(for Western blot analysis) or on coverslips in 24-well plates
(for fluorescence microscopy). Again, include a replicate on a
separate plate for each condition as dark control.
1. Day 1: Start with optoAKT expressing cells on 6-well or
24-well plate transfected/seeded the day before (see
Subheadings 3.1.1 and 3.1.2, respectively), which should have
a confluence of ~80% in the evening. Replace culture medium
with starvation medium (see Note 13) and place plates back
into incubator. It is important that the cells are not exposed to
any source of irradiation (390–480 nm) that would inadvertently excite the optogenetic system during starvation.
2. Day 2: In the morning (~12–15 h post starvation) cells are
ready for the experiment. The exact conditions of irradiation
vary depending on the desired readout.
1. For analysis of optoAKT by Western blotting set the intensity
of the LEDs in the light box to 150 μmol/(m
2
s) and irradiate
the cells 15 times for 5 s/min or constantly with a lower intensity (see Note 14). Intensities higher than 50 μmol/(m
2
s) may
induce photo-cytotoxic effects when applied for extended time
periods. Before further processing the cells ensure that ambient light is turned off and safe light is turned on (see Note 15)
and that there are no additional sources of light which accidentally could activate the optogenetic system.
2. Aspirate medium and wash cells once with PBS before adding
250 μl ice-cold modified TENT cell lysis buffer. Gently shake
the plates to evenly distribute the lysis buffer. Incubate the
plates for 10 min on ice.
3. Use a cell scraper to detach the cells and transfer the cell lysate
into a precooled 1.5 ml Eppendorf tube. Sonicate the lysate for
15 min (30 s/min pulses) at 4 °C to fragment DNA and lower
viscosity. In the next step, centrifuge lysates for 15 min at
10,000 × g and 4 °C to pellet the insoluble components.
3.2 Light Experiment
and Readout
3.2.1 Western Blot
Analysis
Optogenetic Control of Protein Kinases