Biomedical Engineering Reference
In-Depth Information
q
To test effectiveness of negative selection, do not add FIAU to one plate. Carefully label this
plate and expect it to have a much larger number of colonies than those plates with FIAU. This
plate should not be used for picking.
PROTOCOL 10.15
Picking ES Cell Colonies
Equipment and reagents
•
Incubator at 37
◦
Cwith5%CO
2
•
Water bath at 37
◦
C
96 well plate
•
10ml glass pipettes
•
50ml conical tubes
•
15ml conical tubes
•
1000
×
penicillin/gentamicin (0.59% penicillin (w/v)/8% gentamicin (w/v))
•
FBS (heat inactivated)
•
0.25% trypsin with EDTA
•
•
DMEM (high glucose, high bicarbonate, no pyruvate, with
L
-glutamine, Invitrogen, cat.
no. 11965)
•
BME
ESGRO LIF (10
7
units/ml, Chemicon, cat. no. ESG1107)
•
•
G418/geneticin-selective antibiotic (50mg/ml)
•
FIAU (1 mg/ml, dissolved 1 : 1 in ethanol/H
2
O, = 2.7 m
M
for 10 000
×
stock, Moravek
Biochemicals)
•
MEF medium: DMEM, 1 : 1000 penicillin/gentamicin, 10% FBS
ES medium: DMEM, 20% FBS, 1 : 1000 penicillin/gentamicin, 1 : 100 BME,
1 : 10 000 LIF
•
ES medium + selection:
—ESmediumplus:
—1
×
(1 : 10 000 dilution of) FIAU
—1
×
(1 : 167 dilution of) G418 (e.g. 3 ml in 500ml of medium)
•
γ
-irradiated MEF cells plated on 0.1% gelatin-coated plates 48 h prior to ES cell
plating.
•
