Quantitative Flow Cytometric Measurement of Fungal Spore Phagocytosis by Human Phagocytes

Published: January 31, 2024

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 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. Place in a humidified CO2 incubator at 37 °C and in…

Divulgations

The authors have nothing to disclose.

Materials

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

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Quantitative Flow Cytometric Measurement of Fungal Spore Phagocytosis by Human Phagocytes. J. Vis. Exp. (Pending Publication), e21893, doi: (2024).

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