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Quantitative Imaging Flow Cytometry of Legionella-Containing Vacuoles in Dually Fluorescence-Labeled Dictyostelium

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Book cover Legionella

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1921))

Abstract

Legionella pneumophila enters and replicates within protozoan and mammalian phagocytes by forming through a conserved mechanism a specialized intracellular compartment termed the Legionella-containing vacuole (LCV). This compartment avoids fusion with bactericidal lysosomes but communicates extensively with different cellular vesicle trafficking pathways and ultimately interacts closely with the endoplasmic reticulum. In order to delineate the process of pathogen vacuole formation and to better understand L. pneumophila virulence, an analysis of markers of the different trafficking pathways on the pathogen vacuole is crucial. Here, we describe a method for rapid, objective and quantitative analysis of different fluorescently tagged proteins or probes on the LCV. To this end, we employ an imaging flow cytometry approach and use the D. discoideum –L. pneumophila infection model. Imaging flow cytometry enables quantification of many different parameters by fluorescence microscopy of cells in flow, rapidly producing statistically robust data from thousands of cells. We also describe the generation of D. discoideum strains simultaneously producing two different fluorescently tagged probes that enable visualization of compartments and processes in parallel. The quantitative imaging flow technique can be corroborated and enhanced by laser scanning confocal microscopy.

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Abbreviations

ACES:

N-(2-acetamido)-2-aminoethanesulfonic acid

AYE:

ACES yeast extract

Cam:

Chloramphenicol

CYE:

Charcoal yeast extract

DMSO:

Dimethyl sulfoxide

DPBS:

Dulbecco's Phosphate-Buffered Saline

ER:

Endoplasmic reticulum

GFP:

Green fluorescent protein

Icm/Dot:

Intracellular multiplication/defective organelle trafficking

IFC:

Imaging flow cytometry

LCV:

Legionella-containing vacuole

MOI:

Multiplicity of infection

PFA:

Paraformaldehyde

RT:

Room temperature

SSC:

Side scatter

T4SS:

Type IV secretion system

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Acknowledgments

Research in the laboratory of H.H. was supported by the Swiss National Science Foundation (SNF; 31003A_153200), the Novartis Foundation for Medical-Biological Research, and the OPO foundation. A.W. was supported by a grant from the Swedish Research Council (2014-396). Imaging flow cytometry was performed using equipment of the Flow Cytometry Facility (University of Zürich) and microscopy using equipment of the Centre for Microscopy and Image Analysis (University of Zürich).

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Authors and Affiliations

  1. Institute of Medical Microbiology, University of Zürich, Zürich, Switzerland

    Amanda Welin, Stephen Weber & Hubert Hilbi

Authors
  1. Amanda Welin
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  2. Stephen Weber
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  3. Hubert Hilbi
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Corresponding author

Correspondence to Amanda Welin .

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Editors and Affiliations

  1. Institut Pasteur, Biologie des Bactéries Intracellulaires, Paris, France

    Carmen Buchrieser

  2. Institute of Medical Microbiology, University of Zürich, Zürich, Switzerland

    Hubert Hilbi

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Welin, A., Weber, S., Hilbi, H. (2019). Quantitative Imaging Flow Cytometry of Legionella-Containing Vacuoles in Dually Fluorescence-Labeled Dictyostelium. In: Buchrieser, C., Hilbi, H. (eds) Legionella. Methods in Molecular Biology, vol 1921. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9048-1_10

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  • DOI: https://doi.org/10.1007/978-1-4939-9048-1_10

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9047-4

  • Online ISBN: 978-1-4939-9048-1

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