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A RAPID METHOD FOR THE ISOLATION AND COLLECTION OF NUCLEI FROM WHOLE CELL SUSPENSIONS

Authors
Journal
The Journal of Cell Biology
0021-9525
Publisher
The Rockefeller University Press
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Abstract

525.tif A RAPID METHOD FOR THE ISOLATION AND COLLECTION OF NUCLEI FROM WHOLE CELL SUSPENSIONS B. R. A. O'BRIEN. From the School of Anatomy, University of New South Wales, Sydney, N. S. W., Australia INTRODUCTION Thc cytolytic action of certain surfacc-activc agents lcd to the observation that some of the non-ionogcnic cthanoxy compounds lyse the plasma mcmbrane of amphibian erythrocytes and liver parcnchyma cells more rapidly than the nuclear membrane (1). Plasmolysis by these substanccs might provide the basis of a technique for the isolation and collcctlon of nuclei from ccll suspensions that may prove to be morc rapid and lcss damaging than the usual methods which involve the mechanical disruption of the cell. Furthcrmorc, isolation of nuclei by a cytolytic method is casily controlled, may be followed microscopically, and can bc undertaken when the number of cclls involved is rclatlvcly small. The surface-active agent used in this investi- gation was a non-ionogcnic cthanoxy compound- - an iso-octyl phenol condensed with an average of six cthylene oxide groups--known as Nonidct P40 (NP40) and provided by the Shell Co. of Australia, Shell House, Sydney, N. S. W. One advantage of this and allied compounds is their solubility in both aqueous and some non-aqueous mcdia. Thc plasma membrane is lysed in both types of media when NP40 is present, and the rate of lysis can be increased so that the membrane breaks down and the nuclei are l iberated while the cells are being centrifuged through a layer of medium containing NP40 into layers of higher density which do not contain the cytolysing agent. The isolated nuclei may be collected as a pellet or as a layer at a particular density of the suspension medium, METHODS Nucleated crythrocytes from the Queensland cane toad (Bufo floridensis) wcre washed several times and resuspended in ice-cold amphibian Ringer-phosphate solution at pH 7.0. Four methods of isolation wcrc employed. 1

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