title: Reduced Fluorescence versus Forward Scatter Time-of-Flight and Increased Peak versus Integral Fluorescence Ratios Indicate Receptor Clustering in Flow Cytometry.
abstract: Clustering of surface receptors is often required to initiate signal transduction, receptor internalization, and cellular activation. To study the kinetics of clustering, we developed an economic high-throughput method using flow cytometry. The quantification of receptor clustering by flow cytometry is based on the following two observations: first, the fluorescence signal length (FL time-of-flight [ToF]) decreases relative to the forward scatter signal length (FSc-ToF), and second, the peak FL (FL-peak) increases relative to the integral FL (FL-integral) upon clustering of FL-labeled surface receptors. Receptor macroclustering can therefore be quantified using the ratios FL-ToF/FSc-ToF (method ToF) or FL-peak/FL-integral (method Peak). We have used these methods to analyze clustering of two immune receptors known to undergo different conformational and oligomeric states: the BCR and the complement receptor 3 (CR3), on murine splenocytes, purified B cells, and human neutrophils. Engagement of both the BCR and CR3, on immortalized as well as primary murine B cells and human neutrophil, respectively, resulted in decreased FL-ToF/FSc-ToF and increased FL-peak/FL-integral ratios. Manipulation of the actin-myosin cytoskeleton altered BCR clustering which could be measured using the established parameters. To confirm clustering of CR3 on neutrophils, we applied imaging flow cytometry. Because receptor engagement is as a biological process dependent on cell viability, energy metabolism, and temperature, receptor clustering can only be quantified by gating on viable cells under physiological conditions. In summary, with this novel method, receptor clustering on nonadherent cells can easily be monitored by high-throughput conventional flow cytometry.
authors.affiliation: Division of Molecular Immunology, Department of Internal Medicine 3, University of Erlangen-Nuremberg, Nikolaus Fiebiger Centre, 91054 Erlangen, Germany; Division of Genetics and Molecular Medicine, King's College London, Guy's Hospital, London SE1 9RT, United Kingdom; Division of Immunology, Infection and Inflammatory Disease, King's College London, Guy's Hospital, London SE1 9RT, United Kingdom; Division of Genetic Epidemiology, Medical University Innsbruck, 6020 Innsbruck, Austria;
authors.forename: Barbara G
authors.initials: BG
authors.lastname: Fürnrohr
authors.affiliation: Division of Molecular Immunology, Department of Internal Medicine 3, University of Erlangen-Nuremberg, Nikolaus Fiebiger Centre, 91054 Erlangen, Germany;
authors.forename: Merle
authors.initials: M
authors.lastname: Stein
authors.affiliation: Division of Genetics and Molecular Medicine, King's College London, Guy's Hospital, London SE1 9RT, United Kingdom; Division of Immunology, Infection and Inflammatory Disease, King's College London, Guy's Hospital, London SE1 9RT, United Kingdom; Department of Rheumatology, University Hospitals Birmingham National Health Service Foundation Trust, Edgbaston, Birmingham B15 2TH, United Kingdom;
authors.forename: Benjamin
authors.initials: B
authors.lastname: Rhodes
authors.affiliation: Flow Cytometry Core Facility, National Institute for Health Research, Guy's and St. Thomas' National Health Service Foundation Trust/King's College London Comprehensive Biomedical Research Centre, Guy's Hospital, London SE1 9RT, United Kingdom;
authors.forename: Prabhjoat S
authors.initials: PS
authors.lastname: Chana
authors.affiliation: Department of Internal Medicine 3, University of Erlangen-Nuremberg, 91054 Erlangen, Germany; and Institute for Clinical Immunology, Department of Internal Medicine 3, University of Erlangen-Nuremberg, 91054 Erlangen, Germany.
authors.forename: Georg
authors.initials: G
authors.lastname: Schett
authors.affiliation: Division of Genetics and Molecular Medicine, King's College London, Guy's Hospital, London SE1 9RT, United Kingdom; Division of Immunology, Infection and Inflammatory Disease, King's College London, Guy's Hospital, London SE1 9RT, United Kingdom;
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Vyse
authors.affiliation: Department of Internal Medicine 3, University of Erlangen-Nuremberg, 91054 Erlangen, Germany; and Institute for Clinical Immunology, Department of Internal Medicine 3, University of Erlangen-Nuremberg, 91054 Erlangen, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Herrmann
authors.affiliation: Division of Molecular Immunology, Department of Internal Medicine 3, University of Erlangen-Nuremberg, Nikolaus Fiebiger Centre, 91054 Erlangen, Germany; dmielenz@molmed.uni-erlangen.de dmielenz@gmx.de.
authors.forename: Dirk
authors.initials: D
authors.lastname: Mielenz
mesh_terms: D008841:Actin Cytoskeleton / Q000737:chemistry / Q000276:immunology; D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology / Q000378:metabolism* / Q000648:ultrastructure; D002232:Carbocyanines / Q000737:chemistry; D002469:Cell Separation; D005434:Flow Cytometry / Q000379:methods*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry; D057166:High-Throughput Screening Assays; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000276:immunology; D016177:Macrophage-1 Antigen / Q000737:chemistry* / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D009504:Neutrophils / Q000276:immunology / Q000378:metabolism* / Q000648:ultrastructure; D061251:Primary Cell Culture; D021381:Protein Transport; D011947:Receptors, Antigen, B-Cell / Q000737:chemistry* / Q000276:immunology; D013194:Staining and Labeling / Q000379:methods
chemical_list: D002232:Carbocyanines; D005456:Fluorescent Dyes; D007140:Immunoglobulin Fab Fragments; D016177:Macrophage-1 Antigen; D011947:Receptors, Antigen, B-Cell; C085321:cyanine dye 5