User guide

Selecting stable transfectants for two-subunit protein
expression,
continued
Selecting stable
transfectants for
two-subunit protein
expression
48 hours after transfection, passage transfected CHO DG44 Cells (cGMP banked)
in complete CD OptiCHO
™
Medium containing 500 µg/mL of Geneticin
®
reagent
to select for stably transfected clones. To passage cells:
1. Determine viable and total cell counts (see page 16).
2. Dilute the cells in pre-warmed complete CD OptiCHO
™
Medium containing
500 µg/mL of Geneticin
®
reagent to give a final cell density of 5 × 10
5
viable
cells/mL.
3. Incubate flasks in a 37°C incubator containing a humidified atmosphere of
8% CO
2
on an orbital shaker platform rotating at 130–135 rpm.
4. Centrifuge cells at 300 × g for 5 minutes, remove the medium by aspiration,
and add fresh medium to the desired final volume every 3–4 days for
14–21 days until cell viability increases to >90% (see Note below). It is not
necessary to centrifuge the cells and re-suspend them in complete fresh
medium if the dilution factor at the time of passage is >2.
5. When the culture reaches >90% viability, maintain it at 3 × 10
5
viable cells/mL
and scale up the culture as needed.
Note
During the selection round, cell viability may drop dramatically (to <10%) due to
the death of untransfected and transiently-transfected cells. To promote optimal
growth of stably transfected cells, maintain cultures as described in Steps 4–5.
32