- gelatinized 60 mm plates
- cES cell media
The desired clone of mES cells must be thawed and
expanded on MEFs several days prior to microinjection. The
cells are usually grown on T-25 flasks or 60 mm plates.
Approximately 4 days before injection:
Cells are brought up on a T-25 flask or other feeder appropriate
Approximately 2 days before injection:
Move cells to 60 mm plates with appropriate feeder cells at
varying concentrations to give you a choice of optimum plates on
injection day (typical cell numbers for plating are 0.4
x106, 0.8 x106, & 1.2
On the microinjection day:
Feed cells with fresh cES cell media 2 - 3 hr prior to
1 hour before drop-off time:
- Aspirate media from plate.
- Rinse and aspirate cells two times with 5 ml PBS.
- Add 1 ml ES trypsin to 60 mm plate.
- Incubate at 37°C for 3 - 5 minutes.
- Add 4 ml cES cell media to counter the trypsin.
- Pipette up and down until colonies are broken into single cell
- Plate cell suspension onto two 60 mm pre-gelatinized dishes for
45 - 60 min. Add cES cell media for a total of 5 ml on each
- After a fair amount of cells have settled on the bottom, gently
remove non-adherent cells with a 10 ml pipette. Label and
save this supernatant in a 15 ml conical tube at 4°C as a
- With 2 ml of fresh cES cell media, 'blow off' loosely adhering
ES cells with pipette and transfer to a 15 ml conical tube.
- Check for single cell suspension by looking at a drop of
media/cells on a hemacytometer. Continue pipetting if
- Place 2 ml of cells on ice and take to the TMESCSR at the
pre-arranged time (usually 10:00 AM).
Source: TMESCSR 4/10