Quantitative Flow Cytometric Measurement of Fungal Spore Phagocytosis by Human Phagocytes

Abstract

Source: Hartung, S., et al., Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry. J. Vis. Exp. (2019)

This video demonstrates a method for quantifying the phagocytosis of FITC-labeled pathogenic fungal spores by human leukocytes. Phagocytosed spores are discerned from cell-adherent spores through counterstaining, followed by flow cytometric analysis.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Phagocytosis Assay

1. Incubate 2 x 10⁶ leukocytes and 4 x 10⁶ FITC-labeled conidia (multiplicity of infection = 2) in 1.5 mL of RPMI + 10% FCS in a 12-well cell culture plate. As controls, include cells only (no conidia) and cells + unlabeled conidia.
2. Place in a humidified CO₂ incubator at 37 °C and incubate for the desired period of time (e.g., 0.5 h, 2 h, or 4 h).
3. After incubation, harvest cells with a cell scraper and put in a 15 mL tube.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Adhesive foil	Brand	701367
Cell culture plate, 12-well	Greiner Bio-one	665180
Cytometer	BD Biosciences		LSR Fortessa II, lasers: 488 nm (blue), 405 nm (violet), 355 nm (UV) and 640 nm (red)
Ethylenediaminetetraacetic acid (EDTA)	Sigma Aldrich	ED3SS-500g	2 mM in PBS
Fluorescein isothiocyanate (FITC)	Sigma Aldrich	F3651-100MG	0.1 mM in Na2CO3 /PBS solution
Phosphate Buffered Saline (PBS)	ThermoFisher Scientific	189012-014	Without Calcium, without Magnesium
RPMI 1640	ThermoFisher Scientific	61870010	RPMI 1640 Medium, GlutaMAX Supplement
Software for data acquisition and analysis	BD Biosciences
V-bottom plate, 96 well	Brand	781601	Untreated surface
Formaldehyde	Carl Roth	PO87.3	Histofix