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Legionella pp 161-177 | Cite as

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

  • Amanda WelinEmail author
  • Stephen Weber
  • Hubert Hilbi
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

Dictyostelium discoideum ImageStream Imaging flow cytometry Legionella pneumophila Endoplasmic reticulum Membrane dynamics Pathogen vacuole Phagocytosis Phagosome Type IV secretion Vesicle trafficking 

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

Notes

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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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Institute of Medical MicrobiologyUniversity of ZürichZürichSwitzerland

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