Fractionation of isolated liver cells after disruption with a nitrogen bomb and sonication.
Hepatocytes from rat liver were prepared by perfusion with collagenase, and rough and smooth microsomes and mitochondria were prepared after cell disruption. By applying 1000 lb/in2 (1 lb/in2 = 6.9 kPa) in a nitrogen bomb followed by decompression, 75% of the cells were disrupted after four consecutive treatments. Intact mitochondria, and rough and smooth microsomes with little contamination were prepared from the homogenate. A more rapid disruption was attained by a short sonication with a low output, thus increasing the efficiency of homogenization. The microsomal subfractions prepared from this homogenate were comparable to those obtained after decompression. Sonication resulted in smooth microsomes, which exhibited a higher contamination with non-microsomal membranes. These, however, were partly removed by additional centrifugation with a discontinuous sucrose gradient containing divalent cations.
D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D008055:Lipids / Q000032:analysis; D008099:Liver / Q000648:ultrastructure*; D008854:Microscopy, Electron; D008862:Microsomes, Liver / Q000032:analysis / Q000648:ultrastructure; D008930:Mitochondria, Liver / Q000032:analysis / Q000648:ultrastructure; D009243:NAD / Q000378:metabolism; D009584:Nitrogen; D011312:Pressure; D011506:Proteins / Q000032:analysis; D012313:RNA / Q000032:analysis; D051381:Rats; D014465:Ultrasonics
D008055:Lipids; D011506:Proteins; D009243:NAD; D012313:RNA; D009584:Nitrogen