Thawing hESCs on Matrigel plate: Difference between revisions
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1. Prepare Matrigel plate 1-2 h before thawing hESCs. If cooled Matrigel plates are used, place the plates at room temperature for at least an hour prior to plating cells. | 1. Prepare Matrigel plate 1-2 h before thawing hESCs. If cooled Matrigel plates are used, place the plates at room temperature for at least an hour prior to plating cells. | ||
Latest revision as of 00:33, 2 June 2012
1. Prepare Matrigel plate 1-2 h before thawing hESCs. If cooled Matrigel plates are used, place the plates at room temperature for at least an hour prior to plating cells.
2. Thaw hES vial at 37°C water bath (with small frozen portion left).
3. Add 5 mL pre-warmed DMEM/F-12 media (37°C) to a conical tube. Transfer cells to the conical tube and wash the vial with another 5 mL of DMEM/F-12 media.
4. Centrifuge at 300g for 5 minutes.
5. Aspirate excess Matrigel working solution in the 35 mm Matrigel plate and add 1 ml of pre-warmed mTeSR1 media to the plate.
6. After centrifuge is completed, discard the media without disturbing the pellet. Resuspend pellet in 1 ml of mTeSR1 media.
7. Add cell suspension to the Matrigel plate in a drop wise manner.
8. Incubate the plate at 37°C incubator. Medium should be changed daily.