Target Discovery and Validation Reviews and Protocols

Keratin Transgenics and Knockouts 233
5. Remove medium, replace with 5 mL of fresh medium containing Pen/Strep, if
necessary, and replate all cells on 25-cm 2 flask.
6. The next day, replace with fresh medium without antibiotics. Routine culture without
antibiotics is strongly recommended.
7. To subculture, aspirate the medium and wash cells with 1 mL of prewarmed trypsin
per 25-cm 2 flask (2 mL/75 cm 2 ).
8. Remove and replace with fresh trypsin and leave in 37°C incubator until cells are
dislodged by gentle agitation. Dislodging happens almost instantly. Do not
overtrypsinize or damage to the cells may result.
9. Add 1 mL of soybean trypsin inhibitor and 4 mL of medium per 1 mL of trypsin
and pipet up and down against flask wall approx 10–20 times (you may vary the
distance between pipet tip and flask wall between 0.5 and 2.5 cm). It is not essential
to obtain a single cell suspension for routine culture, but large cell clumps
must be avoided because they lead to differentiation.
10. Check suspension under microscope before transferring cells into a centrifuge
tube. There should be a uniform size distribution of cells and cell aggregates.
In the meantime, gelatinize new flask(s). Spin cells for 5 min at 250g (at RT),
aspirate off supernatant, and resuspend cell pellet gently in a few milliliters of
medium. Replate at desired density and add 6–8 mL of medium to small flasks
and 25–30 mL to large flasks. A 25-cm 2 flask yields approx 7–10 million cells,
and a 75-cm 2 flask yields approx 25–30 million cells.
3.3. Preparation of Feeder Cells From STO or MEF Cells
Here, we describe routine culture and inactivation of feeder cells. When ES
cells are grown in the presence of feeders, the culture medium containing serum
should be used (refer to freezing of ES cells in Subheading 2. for medium components).
There are two ways to inactivate feeder cells, mitomycin C treatment
and γ-irradiation.
1. Mitomycin C treatment:
a. Add 10 µg/mL mitomycin C to a confluent culture of feeder cells and place in
incubator for 2 to 3 h.
b. Wash the dishes five times with PBS and collect cells by trypsinization (1 to
2 min). Spin cells 3 min at 250g (at RT), discard supernatant, and then resuspend
1 times in medium and respin.
c. Resuspend cells in ES cell medium and count them. Adjust at appropriate density,
e.g., 106 cells/mL and use them. Mitomycin C-treated feeder cells can be
frozen, following the same regimen as described for ES cell freezing.
2. γ-Irradiation:
a. Grow cells to near confluence. Trypsinize cells from 10 to 15 75-cm2 flasks,
spin down 3 min at 250g. Discard supernatant. Resuspend cells in 10–12 mL of
culture medium in a 50-mL plastic tube.