Difference between revisions of "Isolation of Mouse Embryonic Fibroblasts (MEFs)"

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(copied in protocol from sergey's paper)
 
(updated with more details)
 
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From Zolov et al (under review)
 
From Zolov et al (under review)
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==Materials==
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* Collagenase, type II (LS004204, Worthington, US)
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* Clean surgical blades
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* DMEM/PSG/10% FBS
  
 
==Generation of Mouse Primary Fibroblast Cells==  
 
==Generation of Mouse Primary Fibroblast Cells==  
  
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Fibroblasts were prepared from tails and legs of P0 pups. Tissues from individual animals were kept separate, disaggregated with a surgical blade and digested with 500 U/ml type II collagenase (LS004204, Worthington, US) in 5 ml RPMI media (Cat. # 1640, Invitrogen, US) supplemented with 10% FBS, 2 mM L-glutamine and penicillin/streptomycin for 12 hours at 37°C. After incubation, cells were harvested at 200 g for 5 min and plated in 100 mm dishes, in fresh RPMI medium supplemented with 10% FBS, 2 mM L-glutamine and penicillin/streptomycin. Experiments were performed on cells that were passaged 1-3 times.
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# MEFs were prepared from tails and legs of P0 pups.  
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# Disect  out all four limbs plus the tail.  Place one small piece into PCR tube/plate for genotyping
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# Using a clean surgical blade, chop into tiny pieces.
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# Place into 5 mL of collagenase in DMEM/PSG/10% Serum (500U/mL) in a 15 mL falcon tube
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# Incubate in incubator for 12 hours at 37°C.  
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# After incubation, cells were harvested at 200 g for 5 min
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# Wash cells once with warm media, then resuspend in DMEM. 
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# Plate into 2-3 100 mm dishes and change media afer 1d
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# 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

  1. MEFs were prepared from tails and legs of P0 pups.
  2. Disect out all four limbs plus the tail. Place one small piece into PCR tube/plate for genotyping
  3. Using a clean surgical blade, chop into tiny pieces.
  4. Place into 5 mL of collagenase in DMEM/PSG/10% Serum (500U/mL) in a 15 mL falcon tube
  5. Incubate in incubator for 12 hours at 37°C.
  6. After incubation, cells were harvested at 200 g for 5 min
  7. Wash cells once with warm media, then resuspend in DMEM.
  8. Plate into 2-3 100 mm dishes and change media afer 1d
  9. Split when confluent
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