Abstract
A rapid detergent method for the isolation of nuclei from cat brain cortex is described. It involves the homogenization of the tissue in buffered 0.34 M sucrose with the addition of the non-ionic detergent Cemulsol NPT 12 and the subsequent low speed centrifugal sieving of the nuclei through two layers of sucrose (0.68 M and 1.0 M). The final purification is achieved by high speed centrifugation (40,000 g) of the nuclear suspension layered over 1.8 M sucrose. Observations by light microscopy indicate that highly purified and well preserved nuclei are obtained from neurons and glial cells. Electron microscopy reveals some microsomal contaminants adhering to the nuclear membrane. According to an analysis of the nuclear size distribution, a considerable loss of smaller nuclei (10 to 20µ2), mainly from glial cells, occurs during the purification procedure. The action of different detergents is compared, the best results being obtained with Cemulsol NPT 12 or Triton X-100. Chemical analyses of the purified nuclear fraction give the following content expressed in picograms per nucleus: DNA, 6.54; RNA, 2.94; cholesterol, 1.50; and protein, 97.5. The sucrose density gradient centrifugation of nuclei isolated from cat brain cortex shows that their density is equal to or higher than that of 2.2 M sucrose and is thus similar to the density of nuclei from other tissues. The observation of a varying influence of different suspending media on the density of brain cell nuclei resolves the conflicting data in the literature on the density of these nuclei.
Full Text
The Full Text of this article is available as a PDF (836.1 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- CAMMERMEYER J. Astroglial changes during retrograde atrophy of nucleus facialis in mice. J Comp Neurol. 1955 Feb;102(1):133–150. doi: 10.1002/cne.901020107. [DOI] [PubMed] [Google Scholar]
- CHAUVEAU J., MOULE Y., ROUILLER C. Isolation of pure and unaltered liver nuclei morphology and biochemical composition. Exp Cell Res. 1956 Aug;11(2):317–321. doi: 10.1016/0014-4827(56)90107-0. [DOI] [PubMed] [Google Scholar]
- HEALD P. J. Studies on the phosphoproteins of brain: the intracellular localization in brain of a phosphoprotein involved in the metabolic response of cortical slices to electrical stimulation. Biochem J. 1959 Sep;73:132–141. doi: 10.1042/bj0730132. [DOI] [PMC free article] [PubMed] [Google Scholar]
- HELLER I. H., ELLIOTT K. A. Desoxyribonucleic acid content and cell density in brain and human brain tumors. Can J Biochem Physiol. 1954 Sep;32(5):584–592. [PubMed] [Google Scholar]
- HYMER W. C., KUFF E. L. ISOLATION OF NUCLEI FROM MAMMALIAN TISSUES THROUGH THE USE OF TRITON X-100. J Histochem Cytochem. 1964 May;12:359–363. doi: 10.1177/12.5.359. [DOI] [PubMed] [Google Scholar]
- LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
- MANDEL P., BORKOWSKI T., HARTH S., MARDELL R. Incorporation of 32P in ribonucleic acid of subcellular fractions of various regions of the rat central nervous system. J Neurochem. 1961 Nov;8:126–138. doi: 10.1111/j.1471-4159.1961.tb13534.x. [DOI] [PubMed] [Google Scholar]
- O'BRIEN B. R. A RAPID METHOD FOR THE ISOLATION AND COLLECTION OF NUCLEI FROM WHOLE CELL SUSPENSIONS. J Cell Biol. 1964 Mar;20:525–528. doi: 10.1083/jcb.20.3.525. [DOI] [PMC free article] [PubMed] [Google Scholar]
- ROODYN D. B. A survey of metabolic studies on isolated mammalian nuclei. Int Rev Cytol. 1959;8:279–344. doi: 10.1016/s0074-7696(08)62734-5. [DOI] [PubMed] [Google Scholar]
- SANTEN R. J., AGRANOFF B. W. Studies on the estimation of deoxyribonucleic acid in rat brain. Biochim Biophys Acta. 1963 Jun 25;72:251–262. [PubMed] [Google Scholar]
- TSANEV R., MARKOV G. G. Substances interfering with spectrophotometric estimation of nucleic acids and their elimination by the two-wavelength method. Biochim Biophys Acta. 1960 Aug 26;42:442–452. doi: 10.1016/0006-3002(60)90822-2. [DOI] [PubMed] [Google Scholar]