Quantitative Flow Cytometric Measurement of Fungal Spore Phagocytosis by Human Phagocytes

Overview

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

1. 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.
2. Place in a humidified CO₂ incubator at 37 °C and incubate for the desired period of time (e.g., 0.5 h, 2 h, or 4 h).
3. After incubation, harvest cells with a cell scraper and put in a 15 mL tube.
4. Spin for 5 min at 300 x g at room temperature.
5. Collect supernatant for cytokine analysis or discard if not wanted. Resuspend each sample in 100 µL of PBS + 2 mM EDTA.

2. Antibody Staining

1. For each sample, prepare 100 µL of antibody mix, including APC anti-FITC antibody, according to Table 1.
2. Put a 100 µL sample into one well of a 96-well V-bottom plate. Add 150 µL of PBS + 2 mM EDTA for washing.
3. For color compensation, place 1 x 10⁶ cells for each color in further wells of the 96-well V bottom plate. Include a well of cells that is left unstained. Add 150 µL of PBS + 2 mM EDTA for washing.
4. Cover the plate with an adhesive foil.
5. Spin for 5 min at 300 x g at room temperature. Remove the foil.
6. Discard the supernatant by quickly and forcefully inverting the plate only once over the sink or with a disposable paper towel.
   NOTE: Do not repeat or knock the plate on a paper until dry as this will result in a massive loss of cells from the plate.
7. Resuspend cells in the 100 µL antibody mix, and mix well by pipetting.
8. For color compensation, resuspend the respective cells in 100 µL of PBS + 2 mM EDTA and add a single antibody to each well at the same amount used in the antibody mix.
9. Cover with an adhesive foil and incubate for 20 min at room temperature in the dark.
10. Remove the foil. Add 150 µL of PBS + 2 mM EDTA to each well for washing. Cover with an adhesive foil.
11. Spin for 5 min at 300 x g at room temperature. Remove the foil. Discard the supernatant by quickly and forcefully inverting the plate over the sink or a disposable paper towel.
12. Resuspend in 200 µL of PBS + 2 mM EDTA and transfer cells from each well to a separate round bottom tube. Make sure there are no cell clusters in the suspension. Remove clusters otherwise.
    NOTE: Every cluster that is large enough for the eye to see is large enough to potentially clog the cytometer.

3. Flow Cytometry

1. Start the flow cytometer and let it warm up. Start the acquisition software.
2. Create a new experiment and set up and label the samples.
3. Set up parameters (FSC 250, SSC 250) and detectors for fluorophores FITC, APC, BUV395, V500, and PerCP-Cy5.5.
4. Compensation setup
   1. Open compensation setup.
   2. Indicate individual colors.
   3. Using the control cells left unstained or with individual stainings, set the PMT detector voltages to include all events within the scale.
   4. Record at least 10,000 events of each control.
   5. Use the compensation setup to calculate the spillover of fluorophores and apply it to the experiment's cytometer settings.
5. Recording sample data
   1. Display FSC and SSC in the acquisition software and set a gate around leukocytes.
   2. Based on the leukocyte gate, display dot plot SSC/CD45 and gate for CD45⁺ cells to separate from conidia.
   3. Display CD45⁺ cells in a dot plot CD14/CD66b and gate monocytes (CD14⁺) and neutrophils (CD66b⁺) separately.
   4. Display neutrophils in a dot plot anti-FITC/FITC.
   5. Using the sample with unlabeled conidia, set quadrants for anti-FITC and FITC signals, allowing a maximum of 1% of cells in the respective quadrants.
   6. Repeat steps 3.5.4 and 3.5.5 for the monocyte gate.
   7. Record all samples with at least 20,000 events in the leukocyte gate.

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

Materials

Name	Company	Catalog Number	Comments
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