Elucidating Gene Function through Use of Genetically Engineered Mice - Genomics: Essential Methods - page 224

PROTOCOL 10.8 Harvesting of MEF Cells

Equipment and reagents
• Incubator at 37 ◦ Cwith5%CO 2
• Water bath at 37 ◦ C

1 × Dulbecco's 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))
• DMEM (high glucose, high bicarbonate, without pyruvate, with L -glutamine,
Invitrogen)

FBS (heat inactivated, Hyclone) •

• MEF medium: DMEM, 1 × penicillin/gentamicin, 10% FBS.

Method

Perform the following steps using four to five flasks of expanded MEF cells at
atime.

1 Aspirate medium from each 600ml cell culture flask containing MEF
cells.
2 Rinse cells with 10ml PBS.
3 Aspirate PBS and add 3 ml trypsin/EDTA. Place flasks in 37 ◦ C incubator for
5 min, rocking once during this time. e
4 Add 6ml MEF medium to each flask and wash cells from the bottom by pipetting
up and down five times.
5 Transfer cells to a 50 ml conical tube (pool four to five flasks in one conical
tube).
6 Spin cells down (600 g for 3 min) and resuspend in 0.75 ml of MEF medium per
harvested flask. f

Notes
e Notice cells detaching from the bottom of the flask
f Keep the conical tube with pooled cells on ice.