Endocytosis Assays Using Cleavable Fluorescent Dyes

Shifeng Wang; Chun Wan; Galen T Squiers; Jingshi Shen

doi:10.1007/978-1-0716-2209-4_14

Endocytosis Assays Using Cleavable Fluorescent Dyes

Methods Mol Biol. 2022:2473:181-194. doi: 10.1007/978-1-0716-2209-4_14.

Authors

Shifeng Wang^{1

2}, Chun Wan³, Galen T Squiers³, Jingshi Shen⁴

Affiliations

¹ Department of Chinese Medicine Information Science, Beijing University of Chinese Medicine, Beijing, China. shifeng.wang@colorado.edu.
² Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO, USA. shifeng.wang@colorado.edu.
³ Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO, USA.
⁴ Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO, USA. jingshi.shen@colorado.edu.

PMID: 35819767
DOI: 10.1007/978-1-0716-2209-4_14

Abstract

Endocytosis mediates the entry of surface and extracellular cargoes into the cell. In this chapter, we describe assays to quantitively measure the endocytosis of both soluble and transmembrane cargo proteins, taking advantage of cleavable fluorescent dyes labeling cargo proteins or antibodies recognizing cargo proteins. After removing surface-bound fluorescent dye, internalized cargoes are measured using confocal imaging and flow cytometry. We also describe strategies to determine the role of clathrin-mediated endocytosis (CME) in the internalization of a cargo by using a small molecule inhibitor of CME and knockout (KO) of the AAGAB gene, which encodes an essential regulator of CME.

Keywords: AAGAB; Cargo protein; Clathrin-mediated endocytosis; Endocytosis; GLUT4; Membrane trafficking; Transferrin; Transferrin receptor.

Publication types

Research Support, Non-U.S. Gov't
Research Support, N.I.H., Extramural

MeSH terms

Endocytosis* / genetics
Fluorescent Dyes*

Substances

Fluorescent Dyes