1. Plate an appropriate number of cells on a gelatinized 60 mm dish and grow until confluent, feeding daily.  Cells should be passaged without antibiotics prior to submission.
2. Feed with cES cell media 1 – 2 hours prior to trypsinization.
3. Rinse 2 times with 5 ml PBS.
4. Add 2 ml ES cell trypsin.
5. Incubate at 37°C for 3 - 5 minutes.
6. Counter with 4 ml cES cell media.
7. Pipette up and down until colonies are broken into single cell suspension.
8. Transfer total volume of cells to a 15 ml conical tube.
9. Count cells.  Cell count is not critical, but it must be noted if there are more than 5 x 10⁷ cells/ml.
10. Centrifuge at 1000 rpm for 5 minutes. Aspirate media.
11. Re-suspend pellet in 500 µl PBS.
12. Split volume between two 1.5 ml eppendorf tubes.
13. Label with “Rodent PCR Panel”, Investigator, Construct, Clone, Passage Number, Date.
14. Store at -80°C.
15. Contact DAC Comparative Pathology Lab and arrange for a time to drop off the cell samples.

Anne Pate

anne.f.pate@vanderbilt.edu

936-2654

MCN T322

Vials will need to be delivered on dry ice after making arrangements for submission.