Isolation of Macrophage Early and Late Endosomes by Latex Bead Internalization and Density Gradient Centrifugation.

Abstract

Immortalized macrophage lines and primary macrophages display the ability to internalize small latex beads through the endocytic pathway. This protocol describes a simple and robust method for separating endocytic organelles from macrophages on a sucrose gradient, taking advantage of the significantly lower density of the organelles containing latex beads compared with other intracellular organelles. The latex beads are retained in the endosomes as they mature; therefore, harvesting cells at different time points after internalization permits the purification of different organelle fractions, particularly early and late endosomes.