2.3 Cell Culture
Conditions
1. 125 mL up to 1 L polycarbonate shake flasks (Corning).
2. Climo-shaker ISF1-XC CO 2 orbital shaker with 50 mm orbit
(Ku ¨hner).
3. Complete cultivation medium for HEK293-6E: FreeStyle F17
(Gibco, Life Technologies/Thermo Scientific) supplemented
with 7.5 mM glutamine, 0.1% Pluronic-F68, and 25 μg/mL
G418. Prepare by adding the following components to 1 L
fresh F17 medium: 40 mL of 200 mM stock solution of Lglutamine, 0.5 mL of 50 mg/mL G418-solution, and 10 ml of
10% (w/v) Pluronic-F68 (20 g in 200 mL of water and filter
through 0.2 μm). Mix well and store at 4
C.
4. Complete cultivation medium for Hi5 insect cells:
EX-CELL 405.
5. Complete cultivation medium for Sf21 insect cells:
EX-CELL 420.
2.4 Transfection
Reagents
and Additional
Chemicals for Protein
Production
Prepare all solutions using ultrapure water and analytical grade
reagents. All reagents will be sterile filtered and stored at 4
C
(unless otherwise mentioned).
1. 1 mg/mL 25 kDa Polyethylenimine (PEI_25), linear (Polysciences #23966): Dissolve 0.05 g of PEI in 50 mL of water
and heat until it is completely dissolved. Store aliquots at À70
C.
2. Use 15-mL polystyrene tubes for preparing DNA/PEI_25complexes.
3. 20% (w/v) Tryptone N1 (Organotechnie S.A.S., La Courneuve, France): Weigh 100 g Tryptone N1. Make up to
500 mL with water.
4. 300 g/L Glucose: Weigh 150 g glucose and make up to
500 mL with water.
5. 75 mM Valproic acid (20Â). Dissolve 1.2 g of valproic acid
sodium salt (MW 155.19) and make up to 100 mL in F17
medium (supplemented).
6. Guava EasyCyte™ Mini (Merck, Millipore).
7. CASY Cell Counter (Innovatis).
2.5 Biolayer
Interferometry
Prepare all solutions using ultrapure water and analytical grade
reagents. All reagents are sterile filtered and stored at 4
C (unless
otherwise mentioned).
1. 1 mM EZ-Link Sulfo-NHS-LC-LC-Biotin solution (Thermo
Fisher).
2. Zeba spin desalting columns (Thermo Fisher).
3. OctetRed 96 System (Pall, Fortebio).
64
Jens Ko ¨ nig et al.
Conditions
1. 125 mL up to 1 L polycarbonate shake flasks (Corning).
2. Climo-shaker ISF1-XC CO 2 orbital shaker with 50 mm orbit
(Ku ¨hner).
3. Complete cultivation medium for HEK293-6E: FreeStyle F17
(Gibco, Life Technologies/Thermo Scientific) supplemented
with 7.5 mM glutamine, 0.1% Pluronic-F68, and 25 μg/mL
G418. Prepare by adding the following components to 1 L
fresh F17 medium: 40 mL of 200 mM stock solution of Lglutamine, 0.5 mL of 50 mg/mL G418-solution, and 10 ml of
10% (w/v) Pluronic-F68 (20 g in 200 mL of water and filter
through 0.2 μm). Mix well and store at 4
C.
4. Complete cultivation medium for Hi5 insect cells:
EX-CELL 405.
5. Complete cultivation medium for Sf21 insect cells:
EX-CELL 420.
2.4 Transfection
Reagents
and Additional
Chemicals for Protein
Production
Prepare all solutions using ultrapure water and analytical grade
reagents. All reagents will be sterile filtered and stored at 4
C
(unless otherwise mentioned).
1. 1 mg/mL 25 kDa Polyethylenimine (PEI_25), linear (Polysciences #23966): Dissolve 0.05 g of PEI in 50 mL of water
and heat until it is completely dissolved. Store aliquots at À70
C.
2. Use 15-mL polystyrene tubes for preparing DNA/PEI_25complexes.
3. 20% (w/v) Tryptone N1 (Organotechnie S.A.S., La Courneuve, France): Weigh 100 g Tryptone N1. Make up to
500 mL with water.
4. 300 g/L Glucose: Weigh 150 g glucose and make up to
500 mL with water.
5. 75 mM Valproic acid (20Â). Dissolve 1.2 g of valproic acid
sodium salt (MW 155.19) and make up to 100 mL in F17
medium (supplemented).
6. Guava EasyCyte™ Mini (Merck, Millipore).
7. CASY Cell Counter (Innovatis).
2.5 Biolayer
Interferometry
Prepare all solutions using ultrapure water and analytical grade
reagents. All reagents are sterile filtered and stored at 4
C (unless
otherwise mentioned).
1. 1 mM EZ-Link Sulfo-NHS-LC-LC-Biotin solution (Thermo
Fisher).
2. Zeba spin desalting columns (Thermo Fisher).
3. OctetRed 96 System (Pall, Fortebio).
64
Jens Ko ¨ nig et al.