Biomedical Engineering Reference
In-Depth Information
PROTOCOL 10.9
γ
-Irradiation of MEF Cells
Equipment and reagents
•
50ml conical tubes
•
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
•
DMSO (Sigma)
2ml cryotube vials.
•
Method
1 Expose MEF cells harvested in Protocol 10.8 to a dose of 3000 rad (
γ
-rays).
g
2 To aliquot cells for storage, prepare 50ml of MEF medium containing 20% DMSO in a
50ml conical tube.
3 Mix irradiated cells in a 50ml conical tube with an equal volume of MEF medium
containing 20% DMSO.
4 Mix well and immediately aliquot 1ml per cryotube. Mix the stock of cells frequently
during aliquoting.
5 Remove a small aliquot to count the cells.
h
6 Store the cryotubes at
−
80
◦
C overnight in a polystyrene box.
7 The following day, transfer the tubes to liquid nitrogen.
Notes
g
The exact procedure for irradiating cells will depend on the irradiator and the procedures in
the facility housing it.
h
Calculate how many cells will be present in each ml.
PROTOCOL 10.10
Plating
γ
MEF Cells for Culturing ES Cells
Equipment and reagents
•
Incubator at 37
◦
Cwith5%CO
2
•
Water bath at 37
◦
C
