Isolation of intact nuclei of high purity from mouse liver.
Current methods of nuclear isolation from liver disrupt the plasmalemmae via homogenization and separation of the nuclei by high centrifugal force (HCF) through gradients of sucrose or other substances for up to 80 min. The use of HCF for such a long time increases the potential for nuclear damage and degradation by endogenous proteases. We compared four combinations of alterations to classical nuclear isolation methods as follows. Mouse liver was gently crushed through a fine mesh with and without in vivo perfusion with collagenase. The cell suspension was centrifuged at 600 g to remove gross debris and then at moderate centrifugal force (MCF, 16,000 g) or high centrifugal force (HCF, 70,000 g) through sucrose gradients for 30 min. The purity of the isolated nuclei was assessed biologically and morphologically, including analyses of representative marker proteins for nuclei and cytoplasm. The results indicate that MCF and no collagenase provided the highest nuclear integrity and purity, whereas MCF with collagenase is a viable option if priority is given to yield. The method is especially suited for small samples and so should facilitate studies with human liver biopsies and livers from mice, the most widely used species for gene targeting.
Nagata
Takako
T
Lipid Research Laboratory, Department of Veterans Affairs Medical Center, Washington, DC 20422, USA. tnagata17@gmail.com
Redman
Robert S
RS
Lakshman
Raj
R
D000818:Animals; D001707:Biopsy, Needle; D002467:Cell Nucleus / Q000378:metabolism; D002498:Centrifugation; D003584:Cytological Techniques / Q000191:economics / Q000379:methods*; D006801:Humans; D008099:Liver / Q000166:cytology* / Q000473:pathology; D051379:Mice; D008810:Mice, Inbred C57BL; D013997:Time Factors