One vial of ES cells should be thawed onto one well of a 6-well plate or one 35-mm dish. The plate should be prepared with feeders ahead of time. It is best to lay down the feeders the day before. Although it is NOT optimal, it is possible to have laid the feeders 2 hours before thawing the ES cells.
1. Prepare a 15 ml conical tube with 3-4 mls of media before thawing cells.
2. Thaw vial by placing in a 37-degree water bath.
3. When the ice crystal has thawed immediately pipette the cells into the media in the 15ml conical. Pipette gently 2-3X to dilute the DMSO from the ES cells and place in centrifuge.
4. Spin cells for 5 min. at 1000 rpms.
5. Discard supernatant and add ES media. (For one well in a 6-well plate or a 35-mm plate add 2mls of ES media). Disperse into a single cell suspension.
6. Remove media from well containing pre-plated feeders and add ES cells. (After plating cells, when placing wells into the incubator it is helpful to tilt the dish gently back and forth to prevent cells from clumping in the middle of the dish.)
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