Passaging hESCs on Matrigel plate (HUES)

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1. Prepare Matrigel plate 1-2 h before passaging hESCs. Aspirate excess Matrigel working solution in the 35 mm Matrigel plate and add 1 ml of mTeSR1 media to the plate.

2. Pre-warm mTeSR1 media, DMEM/F-12 basal media and dispase at 37°C.

3. Aspirate media from the hESC culture and wash the plate with 1X PBS.

4. Aspirate PBS and add 500 ul Dispase (for single cell suspension)/ collagenase IV (for clumps) to the plate and incubate at 37°C for 7 minutes.

5. Observe the cells under microscope. Edges of the colonies should start to curl.

6. Add 2 ml of DMEM/F-12 basal media and pipette up and down to detach the cells completely.

7. Transfer cell suspension to a 15 ml conical tube, add 1X PBS to 10 ml and centrifuge at 300g, 5 minutes.

8. Aspirate PBS and resuspend pellet in n ml (n = number of plates to seed) of mTeSR1 media.

9. Add cell suspension to the Matrigel plates in a drop wise manner.

10. Incubate the plate at 37°C incubator. Medium should be changed daily.