Reagents, supplies, and equipment:

• gelatinized 60 mm plates
• PBS
• trypsin
• cES cell media
• ice

Procedure:

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 surface.

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 x10⁶).

On the microinjection day:

Feed cells with fresh cES cell media 2 - 3 hr prior to trypsinization.

1 hour before drop-off time:

1. Aspirate media from plate.
2. Rinse and aspirate cells two times with 5 ml PBS.
3. Add 1 ml ES trypsin to 60 mm plate.
4. Incubate at 37°C for 3 - 5 minutes.
5. Add 4 ml cES cell media to counter the trypsin.
6. Pipette up and down until colonies are broken into single cell suspension.
7. 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 plate.
8. 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 last-resort back-up.
9. With 2 ml of fresh cES cell media, 'blow off' loosely adhering ES cells with pipette and transfer to a 15 ml conical tube.
10. Check for single cell suspension by looking at a drop of media/cells on a hemacytometer. Continue pipetting if necessary.
11. 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