Tina:General cell culture protocols: Difference between revisions
>TinaLo (Created page with '==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 …') |
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===Preparing Matrigel Solution=== | ===Preparing Matrigel Solution=== | ||
1. Thaw | 1. Thaw the stock Matrigel overnight (at 4°C). | ||
2. Add | 2. Add cold DMEM/F12 (1:1) 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) | 3. Aliquot 500 ul of Matrigel solution to a 15 ml conical tubes. (Store at -20°C) | ||
* It is critical to keep everything cold while handling Matrigel. | |||
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1. Thaw a 500 ul aliquot of Matrigel at 4°C for 1-2 h before use. | 1. Thaw a 500 ul aliquot of Matrigel at 4°C for 1-2 h before use. | ||
2. Add 7 ml of DMEM/F12 ( | 2. Add 7 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 Matrigel working solution to a 35 mm culture dish and rock gently to distribute the solution evenly. | ||
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===Thawing hESCs on Matrigel plate=== | ===Thawing hESCs on Matrigel plate=== | ||
1. | 1. Prepare Matrigel plate 1-2 h before thawing hESCs. | ||
2. | 2. Add 5 ml pre-warmed mTeSR1 media (37°C) to a conical tube. | ||
3. | 3. Thaw hES vial at 37°C water bath (with small frozen portion left). | ||
4. | 4. Trans cells to the conical tube and wash the vial with an extra 1 ml of hES media. | ||
5. | 5. Centrifuge at 300g for 5 minutes. | ||
6. | 6. Aspirate excess Matrigel working solution in the 35 mm Matrigel plate and add 1 ml of mTeSR1 media to the plate. | ||
7. Add cell suspension to the Matrigel plate in a drop wise manner. | 7. After centrifuge is completed, discard the media without disturbing the pellet after that. Resuspend pellet in 1 ml of hES media. | ||
(Try to maintain cell aggregates during this process!) | |||
8. Add cell suspension to the Matrigel plate in a drop wise manner. | |||
8. Incubate the plate at 37°C incubator. | 8. Incubate the plate at 37°C incubator. Medium should be changed daily. | ||
===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 hES media to the 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 hES media to the plate. | ||
2. Pre-warm | 2. Pre-warm mTeSR1 media, DMEM/F-12 basal media and dispase at 37°C. | ||
3. Aspirate the | 3. Aspirate media from the hESC culture and wash the plate with 1X PBS. | ||
4. Aspirate | 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. | 5. Observe the cells under microscope. Edges of the colonies should start to curl. | ||
6. Add 2 ml of | 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. | 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 media 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. | ||
10. Incubate the plate at 37°C incubator. | 10. Incubate the plate at 37°C incubator. Medium should be changed daily. | ||
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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 hES media to the 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 hES media to the plate. | ||
2. Pre-warm | 2. Pre-warm mTeSR1 media, DMEM/F-12 basal media and dispase at 37°C. | ||
3. Aspirate the | 3. Aspirate media from the hESC culture and wash the plate with 1X PBS. | ||
4. Aspirate | 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. | 5. Observe the cells under microscope. Edges of the colonies should start to curl. | ||
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6. Unwrap a Pasteur pipette and scrape the colonies off. | 6. Unwrap a Pasteur pipette and scrape the colonies off. | ||
7. Add 2 ml of | 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. | 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 media 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. | ||
11. Incubate the plate at 37°C incubator. | 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 |
Revision as of 01:02, 5 April 2012
Feeder independent hES cells culture
Preparing Matrigel Solution
1. Thaw the stock Matrigel overnight (at 4°C).
2. Add cold DMEM/F12 (1:1) 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)
- It is critical to keep everything cold while handling Matrigel.
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 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.
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. Prepare Matrigel plate 1-2 h before thawing hESCs.
2. Add 5 ml pre-warmed mTeSR1 media (37°C) to a conical tube.
3. Thaw hES vial at 37°C water bath (with small frozen portion left).
4. Trans cells to the conical tube and wash the vial with an extra 1 ml of hES media.
5. Centrifuge at 300g for 5 minutes.
6. Aspirate excess Matrigel working solution in the 35 mm Matrigel plate and add 1 ml of mTeSR1 media to the plate.
7. After centrifuge is completed, discard the media without disturbing the pellet after that. Resuspend pellet in 1 ml of hES media. (Try to maintain cell aggregates during this process!)
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 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 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 media 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 media 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