Difference between revisions of "Isolation of Mouse Embryonic Fibroblasts (MEFs)"
From Bridges Lab Protocols
Davebridges (Talk | contribs) (copied in protocol from sergey's paper) |
Davebridges (Talk | contribs) (updated with more details) |
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From Zolov et al (under review) | From Zolov et al (under review) | ||
+ | |||
+ | ==Materials== | ||
+ | * Collagenase, type II (LS004204, Worthington, US) | ||
+ | * Clean surgical blades | ||
+ | * DMEM/PSG/10% FBS | ||
==Generation of Mouse Primary Fibroblast Cells== | ==Generation of Mouse Primary Fibroblast Cells== | ||
− | + | # MEFs were prepared from tails and legs of P0 pups. | |
+ | # Disect out all four limbs plus the tail. Place one small piece into PCR tube/plate for genotyping | ||
+ | # Using a clean surgical blade, chop into tiny pieces. | ||
+ | # Place into 5 mL of collagenase in DMEM/PSG/10% Serum (500U/mL) in a 15 mL falcon tube | ||
+ | # Incubate in incubator for 12 hours at 37°C. | ||
+ | # After incubation, cells were harvested at 200 g for 5 min | ||
+ | # Wash cells once with warm media, then resuspend in DMEM. | ||
+ | # Plate into 2-3 100 mm dishes and change media afer 1d | ||
+ | # Split when confluent |
Latest revision as of 15:50, 5 September 2012
From Zolov et al (under review)
Materials
- Collagenase, type II (LS004204, Worthington, US)
- Clean surgical blades
- DMEM/PSG/10% FBS
Generation of Mouse Primary Fibroblast Cells
- MEFs were prepared from tails and legs of P0 pups.
- Disect out all four limbs plus the tail. Place one small piece into PCR tube/plate for genotyping
- Using a clean surgical blade, chop into tiny pieces.
- Place into 5 mL of collagenase in DMEM/PSG/10% Serum (500U/mL) in a 15 mL falcon tube
- Incubate in incubator for 12 hours at 37°C.
- After incubation, cells were harvested at 200 g for 5 min
- Wash cells once with warm media, then resuspend in DMEM.
- Plate into 2-3 100 mm dishes and change media afer 1d
- Split when confluent