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mouse keratinocytes, need help

Manfred Blessing Blessing at MZDMZA.ZDV.UNI-MAINZ.DE
Sat Feb 22 05:33:05 EST 1997


Hi, this is christiane from germany

I try to culture primary mouse keratinocytes. Until now i get some on a
5 ml tissue culture dish, but not enough for a successfull culture,
normally after a week, there are only fibroblasts left.
I isolate them from newborn fvb-mice. I incubate the skin on dispase (5
mg/ml) overnight at 4°C, the next day i divide the dermis from the
epidermis and incubate the epidermis in trypsin/EDTA (0,25%/0,1%) for 20
min at 37°C. I stop the trypsin with dialyzed bovine serum and culture
them in MCDB-Medium supplemented with 0,5 microgramm/ml insulin, 0,05
microgramm/ml hydrocortisone, 0,1 ng/ml EGF, 7,5 microgramm/ml bovine
pituitary extract and 1% dialyzed bovine serum. Can anybody help ? I
would be very happy !



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