Biomedical Engineering Reference
In-Depth Information
•
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
•
•
γ
-irradiated MEF cells plated on gelatinized plates 48 h prior to ES cell plating
•
1
×
Dulbecco's PBS
•
Injection buffer:
—8.3 g/l of DME powder without phenol red, without sodium bicarbonate (Sigma cat.
no. D5030)
—4.5g/l
D
-glucose
—25m
M
HEPES
—584mg/l
L
-glutamine.
Method
1 Plate one 0.1% gelatin-coated 10 cm plate with MEF cells 48 h in advance.
2 Prepare ES cell injection buffer.
3 Plate PCR-positive ES cells, using ES medium without selection, from either a fresh
passage or from frozen stocks.
gg
,
hh
,
ii
4 Allow cells to grow until colonies are large. This must be timed precisely so that the
injections can be performed on a planned day. Often this involves starting 5-7 days
prior to blastocyst injection, with one passage in between plating and the injection
day.
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5 On the day of injection, select one 10 cm plate of cells with the best morphology and
density.
6 Rinse the plate with 5 ml of PBS.
7 Aspirate the PBS and add 3 ml of 0.25% trypsin/EDTA.
8 Add 9ml of ES medium and thoroughly pipette the cells up and down against the
bottom of the plate using a 10ml glass pipette (15 times).
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9 Spin the cells down (3 min at 600
g
) and resuspend in 2ml of injection buffer
supplemented with 5% FBS.
10 Place the cells on ice until blastocyst injection.
Notes
gg
Cells from a fresh passage are preferable, but not necessary.
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If using cells from a frozen stock, it is generally advised to make use of a earlier passage at
which the cells are still reasonably concentrated. This is often the third passage after plating
from
−
80
◦
C.
