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.
All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.
1. Phagocytosis Assay
2. Antibody Staining
3. Flow Cytometry
Table 1: Antibody mix for flow cytometry. Amounts of each reagent are given as microliters per sample (1 x 106 cells) to be analyzed.
Reagent | µl per sample |
CD45 BUV395 | 1 |
CD14 V500 | 0.5 |
CD66b PerCP-Cy5.5 | 1.5 |
anti-FITC APC | 0.5 |
PBS + 2 mM EDTA | 97 |
Total | 100 |
The authors have nothing to disclose.
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 |