A gentle method for preparing cyto- and nucleo-skeletons and associated chromatin.
We describe a method for permeabilizing and extracting cells that preserves both structure and function whilst allowing the cell derivatives to be handled freely. Cells are encapsulated in microbeads of agarose; the coat of agarose, which is freely permeable to small molecules, forms a protective layer around fragile cell constituents. Cells are then permeabilized by the non-ionic detergent Triton X-100 or antibody and complement in a buffer whose ionic composition mimics that of the cytoplasm. The resulting structures have been characterized morphologically (by immunofluorescence and electron microscopy) and biochemically. Lysis with Triton removes both cell and nuclear membranes, and extracts most of the cytoplasm to leave chromatin surrounded by cytoskeleton; nucleus and cytoplasm then become accessible to triphosphates, enzymes and antibodies. Lysis with complement permeabilizes the cell membrane but leaves the nuclear membrane intact; triphosphates and restriction enzymes, but not antibodies, can then enter both nucleus and cytoplasm. Both types of lysis yield preparations whose chromatin template remains essentially intact, and which is replicated and transcribed at rates close to, or greater than, those found in vivo. Treatment of complement-lysed cells with Triton reduces the very efficient DNA synthesis, implying that the nuclear membrane is involved, directly or indirectly, in replication.
Jackson
D A
DA
Sir William Dunn School of Pathology, Oxford, UK.
Yuan
J
J
Cook
P R
PR
D002467:Cell Nucleus / Q000648:ultrastructure*; D002843:Chromatin / Q000302:isolation & purification*; D003584:Cytological Techniques; D003599:Cytoskeleton / Q000648:ultrastructure*; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D008854:Microscopy, Electron
D002843:Chromatin