Protocol

Labeling Pinocytotic Vesicles and Cytoplasm with Fluorescent Dextrans or Ficolls for Imaging

Adapted from Basic Methods in Microscopy (eds. Spector and Goldman). CSHL Press, Cold Spring Harbor, NY, USA, 2006.

INTRODUCTION

The eukaryotic cell has evolved to compartmentalize its functions and transport various metabolites among cellular compartments. Therefore, in cell biology, the study of organization and structure/function relationships is of great importance. Dextran, a high-molecular-weight poly-D-glucose, and ficoll, a synthetic polymer of epichlorhydrin and sucrose, are electroneutral, hydrophilic polysaccharides whose size can be varied. This variability in size coupled with their membrane impermeability can make them useful for studying fluid-phase pinocytosis, the size of membrane pores (e.g., nuclear membrane pores), or the environmental conditions and size of a cell compartment. This article presents a method for labeling live or fixed cells with substituted forms of dextran or ficoll attached to a fluorescent conjugate. In the event that the desired probe is not available commercially, this article also provides a method for covalently attaching a fluorophore to substituted polysaccharide.