Biomedical Engineering Reference
In-Depth Information
Method
1 Pre-warm MEF media in a 37
◦
C water bath.
2 Aspirate medium from 600ml flasks and gently rinse each flask with 10ml PBS.
3 Aspirate PBS and add 3 ml 0.25% trypsin/EDTA to each flask. Rock the flask to cover
the entire bottom surface.
4 Incubate at 37
◦
C for 5 min, rocking the flasks twice during the incubation.
d
5 Add 15ml MEF media to each flask and rock well to inhibit trypsin.
6 Dissociate cells by pipetting up and down 10 times with a 10ml glass pipette. When
pipetting down, press the pipette tip to the bottom of the Petri dish to help dissociate
tissue pieces.
7 Transfer cell suspension to a 50ml conical tube.
8 Spin for 3 min at 600
g
.
9 While spinning, aliquot 25ml MEF medium to new 600ml flasks.
10 Resuspend MEF cell pellet from each flask in 15ml MEF medium.
11 Add 5ml to each new 600ml flask containing 25 ml MEF medium (for a 1 : 3 passage).
These are P1 MEF cells. Incubate at 37
◦
C/5% CO
2
.
12 Monitor growth of the P1 cells, and change medium every 2 days until they are 100%
confluent.
Notes
d
Notice cells detaching from the bottom of the flask.
PROTOCOL 10.7
Harvesting and Cryopreservation of P2 MEF Cells
Equipment and reagents
•
Incubator at 37
◦
Cwith5%CO
2
•
Water bath at 37
◦
C
1
×
Dulbecco's PBS
•
0.25% trypsin with EDTA
•
10ml glass pipettes
•
50ml conical tubes
•
15ml conical tubes
•
600ml cell culture flasks with air filter caps (Nunc/Nalgene), coated with 0.1% gelatin
•
1000
×
penicillin/gentamicin (0.59% penicillin (w/v)/8% gentamicin (w/v))
•
