Quantitative Flow Cytometric Measurement of Fungal Spore Phagocytosis by Human Phagocytes
Transcript
Take a multi-well plate containing a human leukocyte suspension. Add FITC-labeled pathogenic fungal spores.
During incubation, the spores swell and break their rigid hydrophobic protein layer, exposing the cell wall polysaccharides recognized by specific pattern recognition receptors on phagocytic cells.
This binding triggers intracellular signaling pathways, leading to actin rearrangement and spore internalization within phagosomes.
Depending on the activation state of phagocytes, some spores remain adhered to the cells without internalization.
Post-incubation, harvest the cells and transfer them to a fresh tube.
Centrifuge. Resuspend the cells in a buffer and transfer them to a multi-well V-bottom plate.
Introduce fluorophore-labeled anti-FITC antibodies that bind to the exposed FITC molecules on adhering spores, counterstaining them.
Centrifuge. Resuspend the cells in a buffer and transfer them to flow cytometer tubes.
Perform flow cytometry to differentiate between internalized and cell-adherent spores.
Phagocytes containing internalized spores display FITC signals, while cells with adherent spores exhibit FITC and counterstain signals.
