Tina:General cell culture protocols: Difference between revisions
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==Feeder independent hES cells culture== | ==Feeder independent hES cells culture== | ||
===Preparing Matrigel | ===Preparing Matrigel Aliquots=== | ||
1. Thaw | 1. Thaw Matrigel stock solution at 4°C, overnight. | ||
2. | 2. Place 15 mL conical tubes and an unopened box of 1000 uL tips at -20°C, overnight. | ||
3. Aliquot | 3. Aliquot 145 uL of Matrigel stock solution to a 15 mL tube as quick as possible. Switch tips every 5 tubes to ensure sterility and that the tips are chilled. | ||
4. Store aliquots at -70°C (for up to 6 months). | |||
* It is critical to keep everything cold while handling Matrigel. | * It is critical to keep everything cold while handling Matrigel. | ||
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===Preparing Matrigel Plates=== | ===Preparing Matrigel Plates=== | ||
1. Thaw a | 1. Thaw a 145 ul aliquot of Matrigel at 4°C for 1-2 h before use. | ||
2. Add | 2. Add 12.5 ml of cold DMEM/F12 (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. | 3. Add 1 ml of the 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. | 4. Leave the plate at RT for 1-2 h or put it at 4°C overnight. | ||
* Matrigel plates are good for | * Matrigel plates are good for 1 weeks at 4°C, although fresh plates are preferable. | ||
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===Thawing hESCs on Matrigel plate=== | ===Thawing hESCs on Matrigel plate=== | ||
1. Prepare Matrigel plate 1-2 h before thawing hESCs. | 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. | 2. Thaw hES vial at 37°C water bath (with small frozen portion left). | ||
3. | 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. | 4. Centrifuge at 300g for 5 minutes. | ||
5. | 5. Aspirate excess Matrigel working solution in the 35 mm Matrigel plate and add 1 ml of pre-warmed mTeSR1 media to the plate. | ||
7. After centrifuge is completed, discard the media without disturbing the pellet. Resuspend pellet in 1 ml of mTeSR1 media. | |||
7. After centrifuge is completed, discard the media without disturbing the pellet. Resuspend pellet in 1 ml of | |||
8. Add cell suspension to the Matrigel plate in a drop wise manner. | 8. Add cell suspension to the Matrigel plate in a drop wise manner. | ||
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===Passaging hESCs on Matrigel plate=== | ===Passaging hESCs on Matrigel plate=== | ||
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 | 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. | 2. Pre-warm mTeSR1 media, DMEM/F-12 basal media and dispase at 37°C. | ||
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7. Transfer cell suspension to a 15 ml conical tube, add 1X PBS to 10 ml and centrifuge at 300g, 5 minutes. | 7. Transfer cell suspension to a 15 ml conical tube, add 1X PBS to 10 ml and centrifuge at 300g, 5 minutes. | ||
8. Aspirate | 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. | 9. Add cell suspension to the Matrigel plates in a drop wise manner. | ||
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8. Transfer cell suspension to a 15 ml conical tube, add 1X PBS to 10 ml and centrifuge at 300g, 5 minutes. | 8. Transfer cell suspension to a 15 ml conical tube, add 1X PBS to 10 ml and centrifuge at 300g, 5 minutes. | ||
9. Aspirate | 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. | 10. Add cell suspension to the Matrigel plates in a drop wise manner. |
Revision as of 01:12, 14 April 2012
Feeder independent hES cells culture
Preparing Matrigel Aliquots
1. Thaw Matrigel stock solution at 4°C, overnight.
2. Place 15 mL conical tubes and an unopened box of 1000 uL tips at -20°C, overnight.
3. Aliquot 145 uL of Matrigel stock solution to a 15 mL tube as quick as possible. Switch tips every 5 tubes to ensure sterility and that the tips are chilled.
4. Store aliquots at -70°C (for up to 6 months).
- It is critical to keep everything cold while handling Matrigel.
Preparing Matrigel Plates
1. Thaw a 145 ul aliquot of Matrigel at 4°C for 1-2 h before use.
2. Add 12.5 ml of cold DMEM/F12 (4°C) to the Matrigel solution tubes. Pipet gently to avoid bubbles formation.
3. Add 1 ml of the 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 1 weeks at 4°C, although fresh plates are preferable.
Thawing hESCs on Matrigel plate
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.
7. After centrifuge is completed, discard the media without disturbing the pellet. Resuspend pellet in 1 ml of mTeSR1 media.
8. 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.
Passaging hESCs on Matrigel plate
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.
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 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.
Cryopreservating hESCs
Freezing media: 90% FBS + 10% DMSO
Osteogenic differentiation media
MEM containing 0-20% heat-inactivated ES-FBS (to be determined), 10−7 M Dexamethasone, 50 μg/ml Ascorbic acid and 10 mM β-glycerolphosphate