Song:MEF Preparation: Difference between revisions

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#Remove the medium, add 10 μg/ml mitomycin-c (diluted in MEF medium) and incubate at 37 °C for 3 h.
#Remove the medium, add 10 μg/ml mitomycin-c (diluted in MEF medium) and incubate at 37 °C for 3 h.
#Remove the mitomycin-c solution from the cells and wash them with DPBS for five times.
#Remove the mitomycin-c solution from the cells and wash them with DPBS for five times.
#Trypsinize the cells, collect in MEF medium, and then centrifuge them.
#Trypsinize the cells, collect in MEF medium, and then centrifuge.
#aliquot into cryovials and freeze at –80 °C.
#Aliquot into cryovials and freeze at –80 °C.
#Refs:[http://www.lifetechnologies.com/us/en/home/references/protocols/cell-culture/stem-cell-protocols/ipsc-protocols/feeder-dependent-culture-hescs.html Numbers for cell culture], [http://www.lifetechnologies.com/us/en/home/references/protocols/cell-culture/stem-cell-protocols/ipsc-protocols/feeder-dependent-culture-hescs.html Feeder density]

Latest revision as of 00:23, 20 September 2013

  1. Grow passage-3(no more than) primary MEFs to confluency.
  2. Remove the medium, add 10 μg/ml mitomycin-c (diluted in MEF medium) and incubate at 37 °C for 3 h.
  3. Remove the mitomycin-c solution from the cells and wash them with DPBS for five times.
  4. Trypsinize the cells, collect in MEF medium, and then centrifuge.
  5. Aliquot into cryovials and freeze at –80 °C.
  6. Refs:Numbers for cell culture, Feeder density