Thaw and passage ES cells at least once before injection.
Cells should be confluent on injection day.
Feed confluent ES cells at least one hour before collection with warm DMEM +15% FBS.
20 minutes before injection, wash surface of dish with PBS, aspirate.
Trypsinize (2 ml for 75 cm2) 3-5 minutes in tissue culture incubator.
Pipet cells to dissociate, collect in 10ml DMEM+15% FBS in a 15ml conical tube.
Pellet 3min., 1000rpm in clinical centrifuge.
Aspirate media.
Tap tube to dissociate cell pellet.
Resuspend in 5-10 ml M2 media (or DMEM).
Pellet 3 min., 1000 rpm
Aspirate.
Tap tube to dissociate cell pellet.
Resuspend cells in 0.5 to 1 ml M2, (or DMEM) depending on size of pellet.
Place tubes on ice for transport.