263
1. Day 1: Begin with a 10-cm plate with ~80% confluent
HEK293T (transfection and lentiviral vector transduction) or
Platinum E (retroviral vector transduction) cells. Pre-warm
culture medium, PBS, and trypsin-EDTA solution in a 37 °C
water bath. When preparing samples for fluorescence microscopy,
place autoclaved coverslips into wells of 24-well plate.
2. Aspirate culture medium and wash once with PBS to remove
residual culture medium before adding 1 ml of trypsin-EDTA
solution. Place cells back into incubator for ~3 min. After incubation use a serological pipette to flush the plate with 5 ml
culture medium and transfer the cell suspension into a 15 ml
conical centrifuge tube. Centrifuge the conical centrifuge tube
containing the cell suspension for 3 min at 300 × g. Afterward,
discard the supernatant and resuspend the cell pellet in 10 ml
culture medium. Dilute appropriate amount of cell suspension
(dependent on CASY setup) in 10 ml CASY ton buffer within
a CASY cup and determine cell number using CASY cell counter
(see Note 3).
1. Seed HEK293T cells with a density of 0.75 × 10
5
cells/cm
2
.
Either diluted in 2 ml culture medium for 6-well plates (for
Western blot analysis) or in 0.5 ml culture medium for 24-well
plates (for fluorescence microscopy). It is important that for
each experimental condition at least one replicate on a separate
plate is prepared, which is going to serve as dark control later.
2. Day 2: Cells should have reached ~60–70% confluence for a
good transfection efficiency. For the transfection of a 6-well
use a total of 3 μg DNA and for 24-well plate a total of 0.75 μg
DNA. Transfect pGR427 and pGR464 in a ratio of 1:1 (see
Note 4). For sole transfection of pGR427 use an empty vector
instead of pGR464 to guarantee comparable transfection conditions. Also include an empty vector control to ensure that
irradiation itself is causing the observed effects.
3. Dilute the DNA in 100 μl or 50 μl opti-MEM for 6-well and
24-well, respectively. Next add PEI solution (9 μl for 6 well;
2.25 μl for 24 well) and vigorously vortex PEI/-opti-MEM/DNA solution for 5 s (see Note 5). Let the solution incubate
for 15 min at room temperature. Prior to applying the solution
pipette it up and down a couple of times to ensure homogeneity then add it dropwise onto cells. Afterward, gently rock the
plate to evenly distribute the PEI/-opti-MEM/-DNA mixture
and put the cells back into the incubator (see Note 6).
4. After 5 h replace the transfection medium with fresh culture
medium and return the cells back into the incubator. After an
incubation period of ~24 h proceed with the experiment (continue with Subheading 3.2).
3.1 Expression
of optoAKT
in Mammalian Cells
3.1.1 Transient
Transfection
Optogenetic Control of Protein Kinases
1. Day 1: Begin with a 10-cm plate with ~80% confluent
HEK293T (transfection and lentiviral vector transduction) or
Platinum E (retroviral vector transduction) cells. Pre-warm
culture medium, PBS, and trypsin-EDTA solution in a 37 °C
water bath. When preparing samples for fluorescence microscopy,
place autoclaved coverslips into wells of 24-well plate.
2. Aspirate culture medium and wash once with PBS to remove
residual culture medium before adding 1 ml of trypsin-EDTA
solution. Place cells back into incubator for ~3 min. After incubation use a serological pipette to flush the plate with 5 ml
culture medium and transfer the cell suspension into a 15 ml
conical centrifuge tube. Centrifuge the conical centrifuge tube
containing the cell suspension for 3 min at 300 × g. Afterward,
discard the supernatant and resuspend the cell pellet in 10 ml
culture medium. Dilute appropriate amount of cell suspension
(dependent on CASY setup) in 10 ml CASY ton buffer within
a CASY cup and determine cell number using CASY cell counter
(see Note 3).
1. Seed HEK293T cells with a density of 0.75 × 10
5
cells/cm
2
.
Either diluted in 2 ml culture medium for 6-well plates (for
Western blot analysis) or in 0.5 ml culture medium for 24-well
plates (for fluorescence microscopy). It is important that for
each experimental condition at least one replicate on a separate
plate is prepared, which is going to serve as dark control later.
2. Day 2: Cells should have reached ~60–70% confluence for a
good transfection efficiency. For the transfection of a 6-well
use a total of 3 μg DNA and for 24-well plate a total of 0.75 μg
DNA. Transfect pGR427 and pGR464 in a ratio of 1:1 (see
Note 4). For sole transfection of pGR427 use an empty vector
instead of pGR464 to guarantee comparable transfection conditions. Also include an empty vector control to ensure that
irradiation itself is causing the observed effects.
3. Dilute the DNA in 100 μl or 50 μl opti-MEM for 6-well and
24-well, respectively. Next add PEI solution (9 μl for 6 well;
2.25 μl for 24 well) and vigorously vortex PEI/-opti-MEM/DNA solution for 5 s (see Note 5). Let the solution incubate
for 15 min at room temperature. Prior to applying the solution
pipette it up and down a couple of times to ensure homogeneity then add it dropwise onto cells. Afterward, gently rock the
plate to evenly distribute the PEI/-opti-MEM/-DNA mixture
and put the cells back into the incubator (see Note 6).
4. After 5 h replace the transfection medium with fresh culture
medium and return the cells back into the incubator. After an
incubation period of ~24 h proceed with the experiment (continue with Subheading 3.2).
3.1 Expression
of optoAKT
in Mammalian Cells
3.1.1 Transient
Transfection
Optogenetic Control of Protein Kinases