Empirical strategy for analytical fractionation of epithelial cells

Abstract

Despite the importance of cell fractionation methods in studies of epithelial transport mechanisms and of a variety of subcellular processes, current practices in cell fractionation have a number of shortcomings. Most cell fractionation studies depend on biochemical markers, but they provide little independent confirmation of the initial assumptions that markers are uniquely associated with particular subcellular structures and that they are uniformly distributed over the surfaces with which they are associated. Moreover, it is generally difficult and time-consuming to design new membrane isolation procedures. After reviewing the analytical nature of physical separation procedures, I suggest an empirical approach to cell fractionation that is both general and comprehensive. This approach uses physical separation procedures to generate spatial distributions of particles in which position is related to such physical properties as sedimentation coefficient, density, cholesterol content, surface charge, and coefficient of partitioning in aqueous polymer two-phase systems. Determination of the frequency distributions of biochemical markers permits detection of separate populations of particles even when the populations lack unique markers. This process requires no a priori assumptions about the subcellular localizations of particles.