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Clinical and Diagnostic Laboratory Immunology, November 2001, p. 1240-1247, Vol. 8, No. 6
1071-412X/01/$04.00+0   DOI: 10.1128/CDLI.8.6.1240-1247.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Early Events in Macrophage Killing of Aspergillus fumigatus Conidia: New Flow Cytometric Viability Assay

Kieren A. Marr,1,2,* Michael Koudadoust,1 Michele Black,1 and S. Arunmozhi Balajee1

Fred Hutchinson Cancer Research Center Program in Infectious Diseases1 and University of Washington Department of Medicine,2 Seattle, Washington

Received 13 April 2001/Returned for modification 17 July 2001/Accepted 13 September 2001

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.

* Corresponding author. Mailing address: Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., D3-100, Seattle, WA 98109. Phone: (206) 667-6702. Fax: (206) 667-4411. E-mail: Kmarr{at}fhcrc.org.

Clinical and Diagnostic Laboratory Immunology, November 2001, p. 1240-1247, Vol. 8, No. 6
1071-412X/01/$04.00+0   DOI: 10.1128/CDLI.8.6.1240-1247.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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