Protocols

Preparation of ES Cells for Injection

Targeted ES cells are thawed and expanded on MEFs prior to microinjection. The cells are usually grown on T-25s 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:

Plate will be moved to 60 mm platess with appropriate feeder cells with varying concentrations to give you a choice of optimum plates on injection day (typical cell numbers will be 0.4 x106, 0.8 x106, & 1.2 x106)

Injection day:

Feed cells with fresh cES Cell Media 2 - 3 hours prior to trypsinization.

Need:

• Gelatinized 60 mm plates
• PBS
• Trypsin
• cES cell media
• Ice

1 hour before drop-off time:

1. Aspirate media for 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 minutes. 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 Transgenic Mouse/ES Cell Shared Resource at the pre-arranged time (usually 10 am).