From owner-cellbiol@net.bio.net Fri Feb 21 22:00:00 1997
Path: biosci!MZDMZA.ZDV.UNI-MAINZ.DE!Blessing
From: Blessing@MZDMZA.ZDV.UNI-MAINZ.DE (Manfred Blessing)
Newsgroups: bionet.cellbiol
Subject: mouse keratinocytes, need help
Date: 22 Feb 1997 02:33:05 -0800
Organization: BIOSCI International Newsgroups for Molecular Biology
Lines: 13
Sender: daemon@net.bio.net
Distribution: world
Message-ID: <330EDCF7.458D@MZDMZA.ZDV.UNI-MAINZ.DE>
NNTP-Posting-Host: net.bio.net

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 !

.