Subpopulations of rat lung fibroblasts with different amounts of type I and type III collagen mRNAs.
A fluorescence-based method using the cell sorter has been devised to separate rat lung fibroblasts into subpopulations. Type I or type III collagen antiserum was used as the primary antibody to react with parent rat lung fibroblasts. This was followed by a fluorescein-conjugated secondary antibody. Specificity of the primary collagen antibody was determined using a monoclonal beta-actin antibody and purified IgG as the primary antibodies. The fluorescent shift of parent rat lung fibroblasts was optimized for the amount of primary collagen antibody and secondary fluorescein-conjugated antibody. An increase in slot blot intensity was observed for pro-alpha 1(I), pro-alpha 2(I), and pro-alpha 1(III) mRNAs with increasing amounts of cellular RNA. When precipitating with type I collagen antibodies, the total cellular steady-state levels of type I procollagen mRNAs were increased in the high intensity cells as compared with the low intensity cells. Alternately, when the type III collagen antibodies were used to precipitate the rat lung fibroblasts, the low intensity cells had increased type I procollagen mRNAs while the high intensity cells had increased type III procollagen mRNA. The subpopulations of rat lung fibroblasts after isolation using the fluorescent cell sorter were readily propagated for at least four passages.
Breen
E
E
Department of Biochemistry, University of Vermont, Burlington 05405.
Falco
V M
VM
Absher
M
M
Cutroneo
K R
KR
D000818:Animals; D000906:Antibodies; D003094:Collagen / Q000235:genetics*; D004274:DNA, Recombinant / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D008168:Lung / Q000166:cytology* / Q000378:metabolism; D008297:Male; D010957:Plasmids; D011347:Procollagen / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D051381:Rats; D011916:Rats, Inbred F344
D000906:Antibodies; D004274:DNA, Recombinant; D011347:Procollagen; D012333:RNA, Messenger; D003094:Collagen