Biomedical Engineering Reference
In-Depth Information
•
DMEM (high glucose, high bicarbonate, without pyruvate, with
L
-glutamine,
Invitrogen)
•
FBS (heat inactivated, Hyclone)
•
MEF medium: DMEM, 1
×
penicillin/gentamicin, 10% FBS
•
Dimethyl sulfoxide (DMSO, Sigma)
•
2ml cryotube vials.
Method
1 Aspirate medium from 600ml flasks and rinse each flask with 10ml PBS.
2 Aspirate PBS and add 3 ml 0.25% trypsin/EDTA to each flask. Rock the flask to cover
the entire bottom surface.
3 Incubate at 37
◦
C for 5 min, rocking the flasks twice during the incubation.
4 Add 8 ml of MEF medium into each flask.
5 Dissociate cells by pipetting up and down 10 times with a 10 ml glass pipette. When
pipetting down, press the pipette tip to the bottom of the Petri dish to help dissociate
tissue pieces.
6 Pool cells from all flasks into 50ml conical tubes.
7 Spin down cells at 600
g
for 5min.
8 During this time, prepare MEF medium containing 20% DMSO.
9 Resuspend cells in 1ml of medium per flask.
10 Add an equal volume of 20% DMSO-MEF medium and mix well.
11 Aliquot cells as quickly as possible to cryotube vials (1 ml per vial) and store in a
polystyrene box at
−
80
◦
C overnight. These are P2 MEF cells.
12 The following day, transfer the tubes to liquid nitrogen for long-term storage.
13 In the future, to avoid
de novo
generation of MEF cells, proceed as follows:
(a) Thaw two vials of P2 MEF cells in a 37
◦
C water bath.
(b) Add cells to 7ml MEF medium in a 15ml tube.
(c) Spin at 600
g
for 3 min.
(d) Aspirate supernatant.
(e) Resuspend pellet in 10ml MEF medium.
(f) Add 5ml cells to the bottom of two 0.1% gelatin-coated 600ml flasks, and add
20ml MEF medium to the bottom of each flask for a total of 25 ml per flask. Mix
well by swirling flask gently.
(g) Place flasks in 37
◦
C incubator. These MEFs are ready to be expanded according to
Protocol 10.6 'Expansion and Passage of MEF cells'. Passage the cells at a 1 : 3 ratio
until they reach P5 (a total of 54 flasks).
