Tina:General cell culture protocols
Feeder independent hES cells culture
Preparing Matrigel Solution
1. Thaw a stock Matrigel (5 ml/bottle) overnight (at 4°C).
2. Add 5 ml of DMEM/F12 (at 4°C) to the Matrigel bottle. Pipet gently to avoid bubbles formation.
3. Aliquot 500 ul of Matrigel solution to a 15 ml conical tubes. (Store at -20°C)
Preparing Matrigel Plates
1. Thaw a 500 ul aliquot of Matrigel at 4°C for 1-2 h before use.
2. Add 7 ml of DMEM/F12 (at 4°C) to the Matrigel solution tubes. Pipet gently to avoid bubbles formation.
3. Add 1 ml of Matrigel working solution to a 35 mm culture dish and rock gently to distribute the solution evenly.
4. Leave the plate at RT for 1-2 h or put it at 4°C overnight.
- Matrigel plates are good for 2 weeks at 4°C, although fresh plates are preferable.
Thawing hESCs on Matrigel plate
1. Add 5 ml pre-warmed hES media (37°C) to a conical tube.
2. Thaw hES vial at 37°C water bath (with small frozen portion left).
3. Trans cells to the conical tube and wash the vial with an extra 1 ml of hES 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 hES media to the plate.
6. After centrifuge is completed, discard the media without disturbing the pellet after that. Re-suspend pellet in 1 ml of hES media.
7. Add cell suspension to the Matrigel plate in a drop wise manner.
8. Incubate the plate at 37°C incubator.
Passaging hESCs on Matrigel plate (HUES)
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 hES media and dispase at 37°C.
3. Aspirate the hES media and wash the plate with 1X PBS.
4. Aspirate the PBS and add 500 ul Dispase? Accutase (for single cell)/ collagenase IV (for colonies) to the plate and incubate at 37°C for 1 min.
5. Observe the cells under microscope. Edges of the colonies should start to curl.
6. Add 2 ml of hES 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 the media and re-suspend pellet in n ml (n = number of plates to seed) of hES media.
9. Add cell suspension to the Matrigel plates in a drop wise manner.
10. Incubate the plate at 37°C incubator.
Passaging hESCs on Matrigel plate mechanically (H1, H9)
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 hES media and dispase at 37°C.
3. Aspirate the hES media and wash the plate with 1X PBS.
4. Aspirate the PBS and add 500 ul collagenase IV to the plate and incubate at 37°C for 1 min.
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 hES 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 the media and re-suspend pellet in n ml (n = number of plates to seed) of hES media.
10. Add cell suspension to the Matrigel plates in a drop wise manner.
11. Incubate the plate at 37°C incubator.