Production of Microcytospheres
Cytoplasmic vesicles can be isolated by several methods which utilize agents with cross-linking capabilities, formaldehyde or glutaraldehyde (Scott et al., 1979). These partially fixed vesicles are unable to attach to a substrate. Since, in certain types of experiments, cytoplasmic vesicles containing only the active ribosomal components and a normal cell membrane are desirable, a method was needed to ensure minimal contamination of vesicles with other organelles. Cytochalasin B has been used in the past to break down the microfilaments associated with the cell membrane (Carter, 1967) and to enucleate cells (Prescott et al., 1971; Veomett et al., 1976; Wigler and Weinstein, 1975; Gopalakrishnan and Tompson, 1975). The resulting cyto-plasts can then be fused with cells or karyoplasts of different origin (Shay, 1977). However, these cytoplasts contain all of the cellular organelles. We describe here a procedure that is speedy, sterile, and applicable to a variety of experiments in which it is essential that cytoplasmic parts of one cell type containing only defined components are fused with other cells. These types of cytoplasmic vesicles are named microcytospheres.
KeywordsVortex Formaldehyde Sucrose Glycerol Citrate
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- Veomett, G., Shay, J. W., Hough, P. V., and Prescott, D. M., 1976, Large-scale enucleation of mammalian cells, in: Methods in Cell Biology, Volume XIII (D. M. Prescott, ed.), Academic Press, New York, pp. 1–6.Google Scholar