2.2.6 Quantification
of the Total Protein
and DNA (Process
Contaminant Estimation)
1. 50-mL centrifuge tubes (Sarstedt, Nu ¨mbrecht, Germany).
2. 2-mL reaction tubes (Sarstedt, Nu ¨mbrecht, Germany).
3. Pipette tips 10, 100, 1000 μL (Sarstedt, Nu ¨mbrecht,
Germany).
4. Piston-operated pipettes 10, 100, and 1000 μL (Eppendorf,
Hamburg, Germany).
5. Pierce™ BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA).
6. PBS (Biochrom, Berlin, Germany).
7. 96-well flat bottom microplate without surface modifications
(Nunc™ Thermo Fisher Scientific, Waltham, MA, USA).
8. Incubator (Binder, Tuttlingen, Germany).
9. Plate reader (BioTek™ Cytation™ 3, BioTek, Winooski,
VT, USA).
10. Deionized water (Milli-Q Academic, Q-Gard
® 1, Merck
KGaA, Darmstadt, Germany).
11. Quant-iT™ PicoGreen
® dsDNA kit (Thermo Fisher Scientific,
Waltham, MA, USA).
12. Black 96-well, flat bottom microplate (Corning, Corning,
New York, USA).
3 Methods
All methods described using the equipment and reagents stated
above. Application of comparable devices and chemicals from alternative different manufacturers is possible.
3.1 Upstream
Processing Based
on an Example
of Vero Cells
3.1.1 Thawing Cells
1. Prepare prewarmed (37
C) DMEM-HG growth medium,
supplemented with 10% (v/v) fetal bovine serum (FBS),
10 mM HEPES, and 4 mM L-glutamine.
2. Thaw cells in the cryovial rapidly in a 37
C water bath (see
Note 2).
3. Sterilize the cryovial with 70% (v/v) ethanol and work under
aseptic conditions from this point on.
4. Using a 2 mL pipette, transfer 1 mL prewarmed complete
growth medium into the cryovial and gently mix the cells
thoroughly by pipetting up and down.
5. Transfer the 2 mL cell suspension into 9 mL prewarmed complete growth medium in a 15 mL centrifuge tube.
6. Centrifuge the cells at 300 Â g, 5 min, RT.
7. Remove and discard the supernatant (residual DMSO).
230
Keven Lothert et al.
of the Total Protein
and DNA (Process
Contaminant Estimation)
1. 50-mL centrifuge tubes (Sarstedt, Nu ¨mbrecht, Germany).
2. 2-mL reaction tubes (Sarstedt, Nu ¨mbrecht, Germany).
3. Pipette tips 10, 100, 1000 μL (Sarstedt, Nu ¨mbrecht,
Germany).
4. Piston-operated pipettes 10, 100, and 1000 μL (Eppendorf,
Hamburg, Germany).
5. Pierce™ BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA).
6. PBS (Biochrom, Berlin, Germany).
7. 96-well flat bottom microplate without surface modifications
(Nunc™ Thermo Fisher Scientific, Waltham, MA, USA).
8. Incubator (Binder, Tuttlingen, Germany).
9. Plate reader (BioTek™ Cytation™ 3, BioTek, Winooski,
VT, USA).
10. Deionized water (Milli-Q Academic, Q-Gard
® 1, Merck
KGaA, Darmstadt, Germany).
11. Quant-iT™ PicoGreen
® dsDNA kit (Thermo Fisher Scientific,
Waltham, MA, USA).
12. Black 96-well, flat bottom microplate (Corning, Corning,
New York, USA).
3 Methods
All methods described using the equipment and reagents stated
above. Application of comparable devices and chemicals from alternative different manufacturers is possible.
3.1 Upstream
Processing Based
on an Example
of Vero Cells
3.1.1 Thawing Cells
1. Prepare prewarmed (37
C) DMEM-HG growth medium,
supplemented with 10% (v/v) fetal bovine serum (FBS),
10 mM HEPES, and 4 mM L-glutamine.
2. Thaw cells in the cryovial rapidly in a 37
C water bath (see
Note 2).
3. Sterilize the cryovial with 70% (v/v) ethanol and work under
aseptic conditions from this point on.
4. Using a 2 mL pipette, transfer 1 mL prewarmed complete
growth medium into the cryovial and gently mix the cells
thoroughly by pipetting up and down.
5. Transfer the 2 mL cell suspension into 9 mL prewarmed complete growth medium in a 15 mL centrifuge tube.
6. Centrifuge the cells at 300 Â g, 5 min, RT.
7. Remove and discard the supernatant (residual DMSO).
230
Keven Lothert et al.