title: Flow cytometry analysis of receptor internalization/shedding.
abstract: Differentiation, proliferation, and chemotaxis responses stem from biological programs that recognize checkpoints at the level of membrane receptor internalization and shedding. Therefore, receptor trafficking represents a crucial regulator of cell functions.
  
  
  
  Here, we present a survey of analyses of receptor internalization vs. shedding based on simple flow cytometry-based techniques. A relevant basic observation is that a fluorochrome-bearing antibody bound to a specific receptor that is translocated from the membrane to the cytoplasm continues to emit light, i.e., the cell remains equally positive for that marker even if the receptor is strongly downregulated or no longer detectable on the membrane. In contrast, fluorescence is lost following receptor shedding.
  
  
  
  The combined uses of standardized hyperosmolar sucrose, acidic treatment and flow cytometry staining at different times allows for fully informative studies of the internalization or shedding pattern of a given receptor. The procedure can be simplified into a straightforward, simple-to-use, and flexible flow cytometry method based on two sequential steps with in-between receptor stimulation. This method obviates the need for time-consuming fluorescence techniques and even confocal microscopy.
  
  
  
  We validate this procedure via comparisons of three receptors, i.e., CXCR4, CD30, and CD25, with membrane trafficking patterns that are involved in biological functions that are relevant to immunity and cancer. © 2016 International Clinical Cytometry Society.
authors.affiliation: Department of Medicine, Section of Hematology, Cancer Research and Cell Biology Laboratory, University of Verona, Verona, Italy.
authors.forename: Antonella
authors.initials: A
authors.lastname: Rigo
authors.affiliation: Department of Medicine, Section of Hematology, Cancer Research and Cell Biology Laboratory, University of Verona, Verona, Italy.
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Vinante
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism; D045744:Cell Line, Tumor; D054377:Chemokine CXCL12 / Q000235:genetics / Q000276:immunology; D004705:Endocytosis / Q000187:drug effects; D005110:Extracellular Space / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D015870:Gene Expression; D006801:Humans; D053645:Interleukin-2 Receptor alpha Subunit / Q000235:genetics / Q000276:immunology*; D017730:Ki-1 Antigen / Q000235:genetics / Q000276:immunology*; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000187:drug effects / Q000276:immunology; D008024:Ligands; D008214:Lymphocytes / Q000187:drug effects / Q000276:immunology* / Q000473:pathology; D009997:Osmotic Pressure; D010799:Phycoerythrin / Q000737:chemistry; D061251:Primary Cell Culture; D011485:Protein Binding; D021381:Protein Transport / Q000187:drug effects; D019718:Receptors, CXCR4 / Q000235:genetics / Q000276:immunology*; D013395:Sucrose / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D000911:Antibodies, Monoclonal; C515255:CXCL12 protein, human; C527276:CXCR4 protein, human; D054377:Chemokine CXCL12; D005456:Fluorescent Dyes; C508017:IL2RA protein, human; D053645:Interleukin-2 Receptor alpha Subunit; D017730:Ki-1 Antigen; D008024:Ligands; D019718:Receptors, CXCR4; D010799:Phycoerythrin; D013395:Sucrose; D016650:Fluorescein-5-isothiocyanate; D013755:Tetradecanoylphorbol Acetate
keywords: CD25; CD30; CXCR4; flow cytometry; receptor internalization; receptor shedding