Fluorescence-activated sorting of rat hepatocytes based on their mixed function oxidase activities towards diethoxyfluorescein.
The formation of ethoxyfluorescein and fluorescein from diethoxyfluorescein by isolated rat hepatocytes has been used as a basis for separating such cells dependent on their mixed function oxidase activities by fluorescence-activated flow cytometry. Five equal fractions defined by computer-generated regions were isolated. Non-viable cells with low fluorescence (region 1) represented 10-15% of the population, while the remainder with higher mixed function oxidase activities (regions 2-5), were greater than 95% viable by Trypan Blue exclusion. In region 1, 30% of the viable cells were binucleate, 67% diploid while in region 5, 13% were binucleate and 69% tetraploid. At 3 h after sorting, following attachment to glass coverslips, exposure of cells to methyl methanesulphonate, retrorsine or norethindrone resulted in unscheduled DNA synthesis which was 2-fold higher in the tetraploid-rich region 5, while aflatoxin B1, benzo[a]pyrene or 2-acetylaminofluorene caused a 5-fold increase in unscheduled DNA synthesis in these cells, relative to the diploid-rich hepatocytes in region 2.
Toxicology Unit, MRC Laboratories, Carshalton, Surrey, U.K.
I N
IN
White
M L
ML
Green
R F
RF
Legg
D000818:Animals; D002273:Carcinogens / Q000494:pharmacology; D002469:Cell Separation; D002851:Chromatography, High Pressure Liquid; D004260:DNA Repair / Q000187:drug effects; D005434:Flow Cytometry / Q000379:methods*; D005453:Fluorescence; D066298:In Vitro Techniques; D008099:Liver / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D008297:Male; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D011003:Ploidies; D051381:Rats; D011916:Rats, Inbred F344
D002273:Carcinogens; D006899:Mixed Function Oxygenases