Early events in macrophage killing of Aspergillus fumigatus conidia: New flow cytometric viability assay

Kieren Marr, M. Koudadoust, M. Black, S. A. Balajee

Research output: Contribution to journalArticle

Abstract

Detailed investigations of macrophage phagocytosis and killing of Aspergillus fumigatus conidia have been limited by technical difficulties in quantifying fungal uptake and viability. In order to study early events in cell pathogen ingestion and killing, we developed a new flow cytometry assay that utilizes the fungus-specific viability dye FUN-1. Metabolically active A. fumigatus conidia accumulate orange fluorescence in vacuoles, while dormant or dead conidia stain green. After incubation within THP-1 cells, recovered conidia are costained with propidium iodide (PI) to discriminate between dormant and dead cells. Flow cytometric measurements of FUN-1 metabolism and PI uptake provide indicators of conidial viability, dormancy, and death. Conidial phagocytosis and killing are also assessed by measurement of green and orange FUN-1 fluorescence within the THP-1 cell population. Compared to previously described methods, this assay has less error introduced by membrane permeability changes and serial dilution of filamentous fungal forms. Results suggest that the THP-1 cells kill conidia rapidly (within 6 h) after exposure. Conidia that are preexposed to human serum are ingested and killed more quickly than are nonopsonized conidia.

Original languageEnglish (US)
Pages (from-to)1240-1247
Number of pages8
JournalClinical and Diagnostic Laboratory Immunology
Volume8
Issue number6
DOIs
StatePublished - 2001
Externally publishedYes

Fingerprint

Aspergillus fumigatus
Fungal Spores
Propidium
Macrophages
Aspergillus
Assays
Coloring Agents
Fluorescence
Flow cytometry
Pathogens
Flow measurement
Fungi
Metabolism
Dilution
Cells
Membranes
Phagocytosis
Vacuoles
Permeability
Flow Cytometry

ASJC Scopus subject areas

  • Microbiology (medical)
  • Immunology and Allergy
  • Clinical Biochemistry
  • Immunology

Cite this

Early events in macrophage killing of Aspergillus fumigatus conidia : New flow cytometric viability assay. / Marr, Kieren; Koudadoust, M.; Black, M.; Balajee, S. A.

In: Clinical and Diagnostic Laboratory Immunology, Vol. 8, No. 6, 2001, p. 1240-1247.

Research output: Contribution to journalArticle

@article{f6519f21ec7041cb992fd426e25bfb90,
title = "Early events in macrophage killing of Aspergillus fumigatus conidia: New flow cytometric viability assay",
abstract = "Detailed investigations of macrophage phagocytosis and killing of Aspergillus fumigatus conidia have been limited by technical difficulties in quantifying fungal uptake and viability. In order to study early events in cell pathogen ingestion and killing, we developed a new flow cytometry assay that utilizes the fungus-specific viability dye FUN-1. Metabolically active A. fumigatus conidia accumulate orange fluorescence in vacuoles, while dormant or dead conidia stain green. After incubation within THP-1 cells, recovered conidia are costained with propidium iodide (PI) to discriminate between dormant and dead cells. Flow cytometric measurements of FUN-1 metabolism and PI uptake provide indicators of conidial viability, dormancy, and death. Conidial phagocytosis and killing are also assessed by measurement of green and orange FUN-1 fluorescence within the THP-1 cell population. Compared to previously described methods, this assay has less error introduced by membrane permeability changes and serial dilution of filamentous fungal forms. Results suggest that the THP-1 cells kill conidia rapidly (within 6 h) after exposure. Conidia that are preexposed to human serum are ingested and killed more quickly than are nonopsonized conidia.",
author = "Kieren Marr and M. Koudadoust and M. Black and Balajee, {S. A.}",
year = "2001",
doi = "10.1128/CDLI.8.6.1240-1247.2001",
language = "English (US)",
volume = "8",
pages = "1240--1247",
journal = "Clinical and Vaccine Immunology",
issn = "1556-6811",
publisher = "American Society for Microbiology",
number = "6",

}

TY - JOUR

T1 - Early events in macrophage killing of Aspergillus fumigatus conidia

T2 - New flow cytometric viability assay

AU - Marr, Kieren

AU - Koudadoust, M.

AU - Black, M.

AU - Balajee, S. A.

PY - 2001

Y1 - 2001

N2 - Detailed investigations of macrophage phagocytosis and killing of Aspergillus fumigatus conidia have been limited by technical difficulties in quantifying fungal uptake and viability. In order to study early events in cell pathogen ingestion and killing, we developed a new flow cytometry assay that utilizes the fungus-specific viability dye FUN-1. Metabolically active A. fumigatus conidia accumulate orange fluorescence in vacuoles, while dormant or dead conidia stain green. After incubation within THP-1 cells, recovered conidia are costained with propidium iodide (PI) to discriminate between dormant and dead cells. Flow cytometric measurements of FUN-1 metabolism and PI uptake provide indicators of conidial viability, dormancy, and death. Conidial phagocytosis and killing are also assessed by measurement of green and orange FUN-1 fluorescence within the THP-1 cell population. Compared to previously described methods, this assay has less error introduced by membrane permeability changes and serial dilution of filamentous fungal forms. Results suggest that the THP-1 cells kill conidia rapidly (within 6 h) after exposure. Conidia that are preexposed to human serum are ingested and killed more quickly than are nonopsonized conidia.

AB - Detailed investigations of macrophage phagocytosis and killing of Aspergillus fumigatus conidia have been limited by technical difficulties in quantifying fungal uptake and viability. In order to study early events in cell pathogen ingestion and killing, we developed a new flow cytometry assay that utilizes the fungus-specific viability dye FUN-1. Metabolically active A. fumigatus conidia accumulate orange fluorescence in vacuoles, while dormant or dead conidia stain green. After incubation within THP-1 cells, recovered conidia are costained with propidium iodide (PI) to discriminate between dormant and dead cells. Flow cytometric measurements of FUN-1 metabolism and PI uptake provide indicators of conidial viability, dormancy, and death. Conidial phagocytosis and killing are also assessed by measurement of green and orange FUN-1 fluorescence within the THP-1 cell population. Compared to previously described methods, this assay has less error introduced by membrane permeability changes and serial dilution of filamentous fungal forms. Results suggest that the THP-1 cells kill conidia rapidly (within 6 h) after exposure. Conidia that are preexposed to human serum are ingested and killed more quickly than are nonopsonized conidia.

UR - http://www.scopus.com/inward/record.url?scp=0034767792&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0034767792&partnerID=8YFLogxK

U2 - 10.1128/CDLI.8.6.1240-1247.2001

DO - 10.1128/CDLI.8.6.1240-1247.2001

M3 - Article

C2 - 11687470

AN - SCOPUS:0034767792

VL - 8

SP - 1240

EP - 1247

JO - Clinical and Vaccine Immunology

JF - Clinical and Vaccine Immunology

SN - 1556-6811

IS - 6

ER -