Tina:General cell culture protocols: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>TinaLo
mNo edit summary
>TinaLo
No edit summary
Line 1: Line 1:
==Feeder independent hES cells culture==
==General ES cell culture==
1. [[mouse ES cell culture medium]]


===Preparing Matrigel Plates===
2. [[mouse ES cell freezing medium]]


1. Thaw a 145 ul aliquot of Matrigel at 4°C for 1-2 h before use.
3. [[human ES cell freezing medium]]


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.
==Feeder dependent ES cell culture==
1. [[Preparation of 0.1% gelatin solution]]


* Matrigel plates are good for 1 weeks at 4°C, although fresh plates are preferable.
2. [[Preparation of gelatin coated plates]]


3. [[MEF culture medium]]


4. [[MEF conditioned medium]]


===Thawing hESCs on Matrigel plate===
5. [[Preparation of inactivated MEFs]]


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.
==Feeder independent hES cell culture (Matrigel)==
1. [[Preparation of Matrigel plates]]


4. Centrifuge at 300g for 5 minutes.  
2. [[Thawing hESCs on Matrigel plate]]


5. Aspirate excess Matrigel working solution in the 35 mm Matrigel plate and add 1 ml of pre-warmed mTeSR1 media to the plate.
3. [[Passaging hESCs on Matrigel plate (HUES)]]


7. After centrifuge is completed, discard the media without disturbing the pellet. Resuspend pellet in 1 ml of mTeSR1 media.
4. [[Passaging hESCs on Matrigel plate mechanically (H1, H9)]]
 
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





Revision as of 00:58, 2 June 2012