Biomedical Engineering Reference
In-Depth Information
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,=2.7mMfor10000
×
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 for 500ml of medium) or 2
×
(1 : 83.5 dilution
of) G418 (during first day of selection)
•
γ
-irradiated MEF cells plated on 0.1% gelatin-coated plates 48 h prior to ES cell
plating
•
1
×
Dulbecco's PBS
•
•
Decay-wave ECM 630 electroporator (BTX - Genetronics)
•
1
×
Hank's balanced salt solution (HBSS)
•
Hemocytometer
•
Trypan blue
Trypsin/EDTA.
•
Method
1 Aspirate ES medium, and rinse plate with 5 ml of PBS.
n
2 Aspirate PBS and add 3 ml of 0.25% trypsin/EDTA. Rock gently until the entire plate is
covered.
