User guide

Transfecting CHO DG44 Cells with FreeStyle
™
MAX Reagent for
single-subunit protein expression,
continued
Transfection
procedure using
pOptiVEC
™
expression
construct
Follow the procedure below to transfect CHO DG44 Cells (cGMP banked) in a
30-mL volume using your pOptiVEC
™
expression construct. We recommend
including negative controls (no DNA, no FreeStyle
™
MAX Reagent) in your
experiment to help you evaluate your results.
1. At 48 hours before transfection, pass CHO DG44 Cells (cGMP banked) at
3 × 10
5
cells/mL in complete CD DG44 Medium. Place the flask(s) on an
orbital shaker platform rotating at 130–135 rpm at 37°C, 8% CO
2
.
2. At 24 hours before transfection, pass CHO DG44 Cells (cGMP banked) at
3 × 10
5
cells/mL in complete CD DG44 Medium. Place the flask(s) on an
orbital shaker platform rotating at 130–135 rpm at 37°C, 8% CO
2
.
3. On the day of transfection, perform a viable cell count (see page 16). To
ensure optimal transfection results, viability of cells must be over 95%.
4. For each transfection or control, transfer 1.5 × 10
7
viable CHO DG44 Cells
(cGMP banked) to a new 125-mL flask. Add pre-warmed, complete CD DG44
Medium to a final volume of 30 mL. Place flask in shaker until ready to
transfect.
Note: Do not centrifuge cells prior to transfection to avoid a decrease in transfection
efficiency.
5. Gently invert the tube of FreeStyle
™
MAX Reagent several times to mix. Do
not vortex.
6. Add 18 µg of plasmid DNA to 600 µL OptiPRO
™
SFM and mix gently. Add
15 µL of FreeStyle
™
MAX Reagent into 600 µL OptiPRO
™
SFM and mix gently.
7. Immediately add diluted FreeStyle
™
MAX Reagent solution to the diluted
DNA solution and mix gently. Incubate the DNA-FreeStyle
™
MAX mix for
10 minutes at room temperature to allow complexes to form. Do not incubate
for longer than 20 minutes.
8. Drop wise add 1.2 mL of DNA-FreeStyle
™
MAX Reagent complex into the
125-mL flask containing cells while slowly swirling the flask.
9. Incubate transfected cell cultures at 37°C, 8% CO
2
on an orbital shaker
platform rotating at 130–135 rpm.
10. At 48 hours post transfection, pass cells into HT-deficient, complete
CD OptiCHO
™
Medium (see page 13). Proceed to Selecting Stable
Transfectants for Single-subunit Protein Expression
, page 52.
48