Wet cleaving of cells: a method to introduce macromolecules into the cytoplasm. Application for immunolocalization of cytosol-exposed antigens.
A new method, called "wet cleaving," has been introduced to allow direct exposure of cytoplasm to externally supplied macromolecules by mechanical rupture of the plasma membrane. Monolayers of adherent cells or poly-L-lysine-attached suspension cells are overlayed with nitrocellulose sheets. By subsequent removal of the sheets, cells are cleaved, thereby exposing the cytoplasm. The method allows bulk quantities of cells to be cleaved in an efficient manner. Cleavage, although imposing some mechanical stress on the cells, leaves most if not all organelles morphologically intact, as shown by electron microscopy. Mechanically ruptured cells are well suited for use in immunocytochemical studies, as is demonstrated with the immunofluorescence localization of vinculin in chicken embryo fibroblasts.
D000818:Animals; D000911:Antibodies, Monoclonal; D002458:Cell Fractionation / Q000379:methods*; D002462:Cell Membrane / Q000648:ultrastructure; D002642:Chick Embryo; D003101:Collodion / Q000494:pharmacology; D003593:Cytoplasm / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D006801:Humans; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D009124:Muscle Proteins / Q000032:analysis; D051381:Rats; D016596:Vinculin
D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; D009124:Muscle Proteins; D016596:Vinculin; D003101:Collodion