Passaging hESCs on Matrigel plate mechanically (H1, H9)

From ZhangLabWiki
Revision as of 00:36, 2 June 2012 by >TinaLo (Created page with "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 hES media to the plate. 2. Pre-w...")
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)
Jump to navigation Jump to search

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 hES 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 collagenase IV to the plate then incubate at 37°C for 1-20 min (to be determined).

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

6. Unwrap a Pasteur pipette and scrape the colonies off.

7. Add 2 ml of DMEM/F-12 basal media to collect cells.

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

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

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

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