Biomedical Engineering Reference
In-Depth Information
13 Allow tissue pieces to settle down for 3-4 min.
14 Transfer supernatant (containing MEF cells) to new 50ml conical tubes.
15 Spin down cells at 600
g
for 5min and aspirate supernatant.
16 Resuspend pellet in 25 ml of MEF medium.
17 Transfer each 25 ml to a 600 ml flask.
18 Mix well by swirling the flasks gently.
19 Place flasks in incubator (37
◦
C/5% CO
2
). These cells are considered to be at passage
zero (P0).
20 Change medium after 24 h and then every 2 days until they are 100% confluent.
21 Monitor cell growth every day for 2-4 days. When cells are 100% confluent they are
ready to be passaged. Protocol 10.6 describes how to expand the MEF cells at a 1 : 3
dilution.
Notes
c
This is a tiring and time-consuming step, but it is very important in order to obtain a good
yield.
PROTOCOL 10.6
Expansion and Passage of 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
•
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))
•
DMEM (high glucose, high bicarbonate, without pyruvate, with
L
-glutamine,
Invitrogen)
•
FBS (heat inactivated, Hyclone)
•
MEF medium: DMEM, 1
×
penicillin/gentamicin, 10% FBS
0.1% gelatin-coated 10 cm Petri dishes.
•
