title: Application of flow cytometry to analyze intracellular location and trafficking of cargo in cell populations.
abstract: Pulse shape analysis (PulSA) is a flow cytometry-based method that involves the measurement of the pulse width and height of a fluorescently labeled molecule simultaneously, enabling a multidimensional analysis of protein localization in a cell at high speed and throughput. We have used the method to detect morphological changes in organelles such as Golgi fragmentation, track protein trafficking from the cell surface, and also discriminate cells with different target protein localizations such as the Golgi, lyso-endosomal network, and the plasma membrane. Here, we describe the basic experimental setup and analytical methods for performing PulSA to examine membrane trafficking processes. We illustrate in particular the application of PulSA for monitoring the trafficking of the membrane-bound enzyme furin and morphological changes to the Golgi caused by Brefeldin A.
authors.affiliation: Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, VIC, 3010, Australia.
authors.forename: Wei Hong
authors.initials: WH
authors.lastname: Toh
authors.forename: Fiona J
authors.initials: FJ
authors.lastname: Houghton
authors.forename: Pei Zhi Cheryl
authors.initials: PZ
authors.lastname: Chia
authors.forename: Yasmin M
authors.initials: YM
authors.lastname: Ramdzan
authors.forename: Danny M
authors.initials: DM
authors.lastname: Hatters
authors.forename: Paul A
authors.initials: PA
authors.lastname: Gleeson
mesh_terms: D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods*; D045683:Furin / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism*; D021381:Protein Transport; D011506:Proteins / Q000378:metabolism*
chemical_list: D011506:Proteins; D045683:Furin