Gentle homogenization conditions should be used to limit possible damage to endosomal elements, particularly when using fluid-phase markers. Clearly, the markers should remain entrapped in vesicles (latent) after homogenization. In addition, harsh conditions should always be avoided to limit the breakage of lysosomes and consequent proteolysis by released hydrolases. Cells grown for 14 to 16 hr are easily homogenized, so each step of the homogenization process should be monitored using a phase-contrast microscope. A balance sheet should be used to follow the distribution of protein and markers during homogenization, flotation, and immunoisolation (see Table 4.3.2 for an example). La
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