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