Quantitative Flow Cytometric Measurement of Fungal Spore Phagocytosis by Human Phagocytes

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.

To begin, transfer 2 million leukocytes and 4 million FITC-labeled conidia in 1.5 milliliters of RPMI supplemented with 10% FCS to a 12-well culture plate. Include a control of cells only with no conidia and a control of cells with unlabeled conidia. Incubate the plate in a humidified carbon dioxide incubator at 37 degrees centigrade for the desired amount of time. When the incubation is complete, use a cell scraper to harvest the cells and transfer them to a 15-milliliter tube.

First, put 100 microliters of each sample and control into one well of a 96-well V-bottom plate. Add 150 microliters of PBS containing 2 millimolar of EDTA for washing. For color compensation, place 1 million cells without conidia for each color in other wells of the plate. Include a well of cells that will be left unstrained. Add 150 microliters of PBS containing 2 millimolar of EDTA to each well for washing.

Next, cover the plate with an adhesive foil and centrifuge at 300 times g at room temperature for five minutes. Then, remove the foil and discard the supernatant by quickly and forcefully inverting the plate only once over either the sink or a disposable paper towel. Resuspend the cells in 100 microliters of antibody mix and mix well by pipetting.

For color compensation, resuspend the respective cells in 100 microliters of PBS containing 2 millimolar of EDTA, and add a single antibody type to each well at the same amount used in the antibody mix. Cover with an adhesive foil, and incubate at room temperature in the dark for 20 minutes.

After this, remove the foil, and add 150 microliters of PBS containing 2 millimolar of EDTA to each well for washing. Cover the plate with adhesive foil again. Centrifuge at 300 times g and at room temperature for five minutes, then, remove the foil, and discard the supernatant by quickly and forcefully inverting the plate over either a sink or a disposable paper towel. Resuspend the cells in 200 microliters of PBS containing 2 millimolar EDTA, and transfer the cell suspension from each well into a separate round-bottom tube.

Start the flow cytometer and let it warm up. In the acquisition software, create a new experiment, and set up and label the new samples. Set up the parameters and the detectors for the appropriate fluorophores as outlined in the text protocol.

In this software, display the appropriate dot plots as outlined in the text protocol. Using the cells-only sample, acquire some cells and set the gate around leukocytes. Based on the leukocyte gate, gate for CD45-positive positive cells to separate from conidia in the SSC/CD45 plot. In a dot plot CD14/CD66b, gate neutrophils and monocytes separately.

After this, change from the cells-only sample to unlabeled conidia. In a dot plot, anti-FITC/FITC display neutrophils, and set quadrants for anti-FITC and FITC signals. Open the statistics view and adjust the quadrants, allowing a maximum of 1% of cells in the quadrants Q1, Q2, and Q4.

Repeat this process for the monocyte gate. In the leukocyte gate, record all samples with at least 20,000 events. Phagocytosis of FITC-labeled conidia by human neutrophils can be read from the statistics views.