Tina:General cell culture protocols: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>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 …')
 
>TinaLo
 
(8 intermediate revisions by the same user not shown)
Line 1: Line 1:
==Feeder independent hES cells culture==
==General ES cell culture medium==
===Preparing Matrigel Solution===
*'''mouse ESCs'''
1. [[mESCs culture medium]]


1. Thaw a stock Matrigel (5 ml/bottle) overnight (at 4°C).
2. [[mESCs freezing medium]]


2. Add 5 ml of DMEM/F12 (at 4°C) to the Matrigel bottle. Pipet gently to avoid bubbles formation.
*'''human ESCs'''
3. [[hESCs culture medium]]


3. Aliquot 500 ul of Matrigel solution to a 15 ml conical tubes. (Store at -20°C)
4. [[hESCs freezing medium]]






===Preparing Matrigel Plates===
==Feeder dependent ESCs culture protocol==
*'''Coating'''
1. [[Preparation of gelatin coated plates]]


1. Thaw a 500 ul aliquot of Matrigel at 4°C for 1-2 h before use.
*'''MEF'''
2. [[MEF culture medium]]


2. Add 7 ml of DMEM/F12 (at 4°C) to the Matrigel solution tubes. Pipet gently to avoid bubbles formation.
3. [[Preparation of inactivated MEFs]]


3. Add 1 ml of Matrigel working solution to a 35 mm culture dish and rock gently to distribute the solution evenly.
*'''mouse ESCs'''
4. [[Thawing mESCs on feeder]]


4. Leave the plate at RT for 1-2 h or put it at 4°C overnight.
5. [[Passaging mESCs on feeder]]


* Matrigel plates are good for 2 weeks at 4°C, although fresh plates are preferable.
*'''human ESCs'''
6. [[Thawing hESCs on feeder]]


7. [[Passaging hESCs on feeder]]


*'''Cryopreservation'''
8. [[Cryopreservation of ESCs]]


===Thawing hESCs on Matrigel plate===
==Feeder independent ESCs culture protocol(Matrigel)==
1. [[Preparation of Matrigel plates]]


1. Add 5 ml pre-warmed hES media (37°C) to a conical tube.
2. [[Preparation of MEF conditioned medium]]


2. Thaw hES vial at 37°C water bath (with small frozen portion left).
3. [[Thawing hESCs on Matrigel plate]]


3. Trans cells to the conical tube and wash the vial with an extra 1 ml of hES media.
4. [[Passaging hESCs on Matrigel plate (HUES)]]


4. Centrifuge at 300g for 5 minutes.  
5. [[Passaging hESCs on Matrigel plate mechanically (H1, H9)]]


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.
==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
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.

Latest revision as of 22:27, 7 May 2013

General ES cell culture medium[edit]

  • mouse ESCs

1. mESCs culture medium

2. mESCs freezing medium

  • human ESCs

3. hESCs culture medium

4. hESCs freezing medium


Feeder dependent ESCs culture protocol[edit]

  • Coating

1. Preparation of gelatin coated plates

  • MEF

2. MEF culture medium

3. Preparation of inactivated MEFs

  • mouse ESCs

4. Thawing mESCs on feeder

5. Passaging mESCs on feeder

  • human ESCs

6. Thawing hESCs on feeder

7. Passaging hESCs on feeder

  • Cryopreservation

8. Cryopreservation of ESCs

Feeder independent ESCs culture protocol(Matrigel)[edit]

1. Preparation of Matrigel plates

2. Preparation of MEF conditioned medium

3. Thawing hESCs on Matrigel plate

4. Passaging hESCs on Matrigel plate (HUES)

5. Passaging hESCs on Matrigel plate mechanically (H1, H9)


Osteogenic differentiation media[edit]

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