Biomedical Engineering Reference
In-Depth Information
•
E13-15 embryos from G418 resistant mouse
•
Isoflurane (Baxter Healthcare)
•
Sterile dissection tools
•
1
×
Dulbecco's phosphate-buffered saline (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))
•
Dulbecco's modified Eagle's medium (DMEM; high glucose, high bicarbonate, without
pyruvate, with
L
-glutamine, Invitrogen)
•
Fetal bovine serum (FBS, heat inactivated, Hyclone)
•
MEF medium: DMEM, 1
×
penicillin/gentamicin, 10% FBS
10 cm diameter Petri dishes.
•
Method
1 Sacrifice the pregnant mouse after isoflurane-induced anesthesia.
2 Dissect out the uterus and place in a large Petri dish with PBS.
3 Use sterile forceps to free the embryos from the uterus and place in a new Petri dish
with PBS.
4 Use two sterile forceps to remove and discard all red organs from the thoracic and
abdominal cavities of each embryo.
5 After dissection of each embryo, place in a new Petri dish with fresh PBS.
6 When all embryos have been dissected, swirl gently to wash.
7 Transfer the embryos to a 10 cm Petri dish containing 5 ml of 0.25%
trypsin/EDTA.
8 Use clean, sterile scissors to chop the embryos into small pieces.
c
9 Add 5ml of 0.25% trypsin and incubate at 37
◦
C for 15min.
10 Dissociate cells by pipetting the mixture up and down with a 10ml glass pipette about
20 times. When pipetting down, press the pipette tip to the bottom of the Petri dish to
help dissociate tissue pieces.
11 Repeat steps 9 and 10 three more times, but reduce the incubation time to 10 min
(37
◦
C/5% CO
2
).
12 Divide the mixture into two 50ml conical tubes and add equal volumes of MEF
medium.
