Isolation of rat Kupffer cells: a combined methodology for highly purified primary cultures.
We report a four-step procedure that optimizes the methodology for isolation of highly purified rat Kupffer cells (KC). We combined the previously reported techniques of enzymatic tissue treatment, density gradient centrifugation, centrifugal elutriation and selective adherence. ED-2 immunophenotyping and non-specific esterase histochemistry were used for cell identification. This combination resulted in a satisfactorily high yield of 80-100 x 10(6)KCs per liver, over 95% positive for ED-2 and 98% viable cells. Cultures of isolated KCs were functionally intact and exhibited a concentration and time-dependent LPS-induced TNF-alpha and nitric oxide production.
Valatas
V
V
Liver Research Laboratory, Faculty of Medicine, University of Crete, P.O. Box 1393, Heraklion, GR-71409 Crete, Greece.
Xidakis
C
C
Roumpaki
H
H
Kolios
G
G
Kouroumalis
E A
EA
D000818:Animals; D002448:Cell Adhesion; D018929:Cell Culture Techniques; D002469:Cell Separation / Q000379:methods*; D007728:Kupffer Cells / Q000166:cytology*; D008297:Male; D010969:Plastics; D051381:Rats; D017207:Rats, Sprague-Dawley; D013047:Specific Pathogen-Free Organisms
D010969:Plastics