Incorporation of Macromolecules into Living Cells

This chapter describes the incorporation of macromolecules into living cells. Techniques for loading impermeant molecules into the cytoplasm of living cells have become essential cell and molecular biological tools. There are several variations of the mechanical strategy for temporarily disrupting plasma membrane or its derivatives. Cells allowed to pinocytose macromolecules in hypertonic medium are loaded by osmotically lysing such pinosomes. Scrape loading is another simple technique for loading cell populations with macromolecules. Adherent cells are scraped off of their substratum with a rubber policeman in the presence of the macromolecules to be loaded. The major variable influencing loading and cell yield from the scrape loading procedure is the strength with which cells adhere to their substratum. Radiolabeled molecules can be used as tracers in quantitative measurements of amount of loading. Phase contrast microscopy provides a rapid way of visually assessing cell health and fluorescence microscopy of qualitatively assessing the extent to which individual cells and the population as a whole are loaded.