260
5. Cell culture-certified disposable plasticware: 10-cm Petri
dishes, 6-well and 24-well plates, serological pipettes, and 15and 50 ml conical centrifuge tubes.
6. Centrifuge with rotor which allows centrifugation of 15 ml
conical centrifuge tubes.
7. CO 2 incubator at 37 °C and 5% CO 2 .
1. Opti-MEM (Thermo Scientific, cat. no. 31985-062). Store at
4 °C.
2. Polyethylenimine (PEI) transfection solution: 1 mg/ml PEI in
H 2 O, adjusted to pH 7.0 with HCl. After sterile filtration (Ø
0.22 μm) 500 μl aliquots are stored at −80 °C. Avoid repeated
freeze-thaw cycles.
3. Silica-based anion-exchange DNA purification kits.
1. Advanced DMEM (Thermo Scientific, cat. no. 12391-015).
2. Cholesterol: 5 mM diluted in PBS.
3. Egg lecithin (Serva Electrophoresis GmbH): 5 mM diluted in
Ethanol.
4. 1× chemical defined lipid concentrate (Gibco, cat. no.
11905031).
5. Hexadimethrine bromide (polybrene): Dilute in deionized
water to a final concentration of 8 mg/ml.
6. Centrifuge that allows centrifugation of cell culture plates.
7. Syringe and 0.45 μm filter (low protein binding).
1. Light boxes: Build out of opaque PVC material. Panels of
LEDs emitting light with a wavelength of 450 nm should be
placed on the top of the box (Roithner LaserTechnik, LED450series). The intensity of the LEDs has to be adjustable. Further,
it is advantageous to include a mean of further programming
the LEDs ON/OFF time to address temporal aspects of signaling. A ventilation of the light box should be included to
ensure that the temperature as well as atmosphere in the box
corresponds to the surroundings (see Note 1).
2. LED safe lights with wavelength longer than 500 nm.
1. 5× SDS-loading buffer: 10% (w/v) SDS, 0.3 M Tris–HCl (pH
6.8), 50% (v/v) Glycerin, 12.5% (v/v) ß-mercaptoethanol,
0.05% (w/v) bromphenol blue in fully desalinated H 2 O.
2. ECL solutions.
3. TENT cell lysis buffer (modified): 20 mM Tris–HCl (pH 8.0),
1 mM EDTA, 100 mM NaCl, 0.5% (v/v) Triton X-100, 1 mM
sodium orthovanadate, 10 mM sodium pyrophosphate,
2.2 Transient
Transfection
2.3 Transduction
2.4 Irradiation
and Readout
2.5 Western Blot
Wignand W.D. Mühlhäuser et al.
5. Cell culture-certified disposable plasticware: 10-cm Petri
dishes, 6-well and 24-well plates, serological pipettes, and 15and 50 ml conical centrifuge tubes.
6. Centrifuge with rotor which allows centrifugation of 15 ml
conical centrifuge tubes.
7. CO 2 incubator at 37 °C and 5% CO 2 .
1. Opti-MEM (Thermo Scientific, cat. no. 31985-062). Store at
4 °C.
2. Polyethylenimine (PEI) transfection solution: 1 mg/ml PEI in
H 2 O, adjusted to pH 7.0 with HCl. After sterile filtration (Ø
0.22 μm) 500 μl aliquots are stored at −80 °C. Avoid repeated
freeze-thaw cycles.
3. Silica-based anion-exchange DNA purification kits.
1. Advanced DMEM (Thermo Scientific, cat. no. 12391-015).
2. Cholesterol: 5 mM diluted in PBS.
3. Egg lecithin (Serva Electrophoresis GmbH): 5 mM diluted in
Ethanol.
4. 1× chemical defined lipid concentrate (Gibco, cat. no.
11905031).
5. Hexadimethrine bromide (polybrene): Dilute in deionized
water to a final concentration of 8 mg/ml.
6. Centrifuge that allows centrifugation of cell culture plates.
7. Syringe and 0.45 μm filter (low protein binding).
1. Light boxes: Build out of opaque PVC material. Panels of
LEDs emitting light with a wavelength of 450 nm should be
placed on the top of the box (Roithner LaserTechnik, LED450series). The intensity of the LEDs has to be adjustable. Further,
it is advantageous to include a mean of further programming
the LEDs ON/OFF time to address temporal aspects of signaling. A ventilation of the light box should be included to
ensure that the temperature as well as atmosphere in the box
corresponds to the surroundings (see Note 1).
2. LED safe lights with wavelength longer than 500 nm.
1. 5× SDS-loading buffer: 10% (w/v) SDS, 0.3 M Tris–HCl (pH
6.8), 50% (v/v) Glycerin, 12.5% (v/v) ß-mercaptoethanol,
0.05% (w/v) bromphenol blue in fully desalinated H 2 O.
2. ECL solutions.
3. TENT cell lysis buffer (modified): 20 mM Tris–HCl (pH 8.0),
1 mM EDTA, 100 mM NaCl, 0.5% (v/v) Triton X-100, 1 mM
sodium orthovanadate, 10 mM sodium pyrophosphate,
2.2 Transient
Transfection
2.3 Transduction
2.4 Irradiation
and Readout
2.5 Western Blot
Wignand W.D. Mühlhäuser et al.