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MEF culture and mESC


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#1 JWChung

JWChung

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Posted 14 October 2010 - 06:00 AM

Hi guys. I previously posted on my problems regarding MEF culture. I've now been able to overcome the problem, and passage up to P7. However, i still need to fine tune my steps as I've not been able to progress beyond P7.
Anyway, I'm moving on to ESC culture, and i used 2 methods. One involve culturing the whole blastocyst, then isolating the cells using trypsin, suck in and out to make single cell, and then culture. Another involve mechanical isolation of inner cell mass using 21G needle, then culturing the cut pieces. What are your opinions on these 2 methods, regarding their ease of use and success rate? Also, i'm actually not really sure whether i'm doing the correct thing, so i would love it if you guys can comment on the photos i'm uploading. Especially regarding the cultured ESC, are they really what i want (stem cells). i'm still waiting for the stains to arrive, so i cant do any staining yet, but i was hoping you guys can give me some idea through visual confimration. Thanks guys

Pictures:
MEF (240910) P2 0.25% 10x 1 = Shows one of my MEF cultures at P2
ESC (Blastocyst Day 0-6) = Shows the culture of whole blastocyst
1_866 = Shows a piece of the outgrowth from blastocyst cultured (1st treatment)
1_868 = Shows a clump of cell, possible ESC (2nd treatment)

Attached Thumbnails

  • MEF (240910) P2 0.25% 10x 1.jpg
  • ESC (Blastocyst, Day 0).jpg
  • ESC (Blastocyst, Day 2).jpg
  • ESC (Blastocyst, Day 5).jpg
  • ESC (Blastocyst, Day 6).jpg
  • 1_866.jpg
  • 1_868.jpg





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