Using Flow Cytometry to Analyze Cryptococcus Infection of Macrophages

  • Robert J. Evans
  • Kerstin Voelz
  • Simon A. Johnston
  • Robin C. May
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1519)

Abstract

Flow cytometry is a powerful analytical technique, which is increasingly being used to study the interaction between host cells and intracellular pathogens. Flow cytometry is capable of measuring a greater number of infected cells within a sample compared to alternative techniques such as fluorescence microscopy. This means that robust quantification of rare events during infection is possible. Our lab and others have developed flow cytometry methods to study interactions between host cells and intracellular pathogens, such as Cryptococcus neoformans, to quantify phagocytosis, intracellular replication, and non-lytic expulsion or “vomocytosis” from the phagosome. Herein we describe these methods and how they can be applied to the study of C. neoformans as well as other similar intracellular pathogens.

Key words

Cryptococcus neoformans Cryptococcus gattii Flow cytometry Macrophage Infection Mycology 

References

  1. 1.
    Feldmesser M, Kress Y, Novikoff P, Casadevall A (2000) Cryptococcus neoformans is a facultative intracellular pathogen in murine pulmonary infection. Infect Immun 68:4225–4237, PMC101732CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Ma H, Croudace JE, Lammas DA, May RC (2006) Expulsion of live pathogenic yeast by macrophages. Curr Biol 16:2156–2160CrossRefPubMedGoogle Scholar
  3. 3.
    Nicola AM, Robertson EJ, Albuquerque P, Derengowski Lda S, Casadevall A (2011) Nonlytic exocytosis of Cryptococcus neoformans from macrophages occurs in vivo and is influenced by phagosomal pH. MBio 2(4). doi:10.1128/mBio.00167,11. Print 2011
  4. 4.
    Alvarez M, Casadevall A (2006) Phagosome extrusion and host-cell survival after Cryptococcus neoformans phagocytosis by macrophages. Curr Biol 16:2161–2165CrossRefPubMedGoogle Scholar
  5. 5.
    Alanio A, Desnos-Ollivier M, Dromer F (2011) Dynamics of Cryptococcus neoformans-macrophage interactions reveal that fungal background influences outcome during cryptococcal meningoencephalitis in humans. MBio 2(4). doi:10.1128/mBio.00158,11. Print 2011
  6. 6.
    Ma H, Hagen F, Stekel DJ, Johnston SA, Sionov E, Falk R et al (2009) The fatal fungal outbreak on Vancouver Island is characterized by enhanced intracellular parasitism driven by mitochondrial regulation. Proc Natl Acad Sci U S A 106:12980–12985, Pmc2722359CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Voelz K, Johnston SA, Rutherford JC, May RC (2010) Automated analysis of cryptococcal macrophage parasitism using GFP-tagged cryptococci. PLoS One 5(12), e15968CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Robert J. Evans
    • 1
    • 2
    • 3
  • Kerstin Voelz
    • 1
  • Simon A. Johnston
    • 2
    • 3
  • Robin C. May
    • 1
  1. 1.Institute of Microbiology and Infection, School of Biosciences, College of Life and Environmental SciencesUniversity of BirminghamBirminghamUK
  2. 2.Bateson Centre, Firth CourtUniversity of Sheffield, Western BankSheffieldUK
  3. 3.Department of Infection, Immunity and Cardiovascular Disease, Medical SchoolUniversity of SheffieldSheffieldUK

Personalised recommendations