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
- 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 incubate for the desired period of time (e.g., 0.5 h, 2 h, or 4 h).
- After incubation, harvest cells with a cell scraper and put in a 15 mL tube.
- Spin for 5 min at 300 x g at room temperature.
- Collect supernatant for cytokine analysis or discard if not wanted. Resuspend each sample in 100 µL of PBS + 2 mM EDTA.
2. Antibody Staining
- For each sample, prepare 100 µL of antibody mix, including APC anti-FITC antibody, according to Table 1.
- 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.
- 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.
- Cover the plate with an adhesive foil.
- Spin for 5 min at 300 x g at room temperature. Remove the foil.
- 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. - Resuspend cells in the 100 µL antibody mix, and mix well by pipetting.
- 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.
- Cover with an adhesive foil and incubate for 20 min at room temperature in the dark.
- Remove the foil. Add 150 µL of PBS + 2 mM EDTA to each well for washing. Cover with an adhesive foil.
- 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.
- 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
- Start the flow cytometer and let it warm up. Start the acquisition software.
- Create a new experiment and set up and label the samples.
- Set up parameters (FSC 250, SSC 250) and detectors for fluorophores FITC, APC, BUV395, V500, and PerCP-Cy5.5.
- Compensation setup
- Open compensation setup.
- Indicate individual colors.
- Using the control cells left unstained or with individual stainings, set the PMT detector voltages to include all events within the scale.
- Record at least 10,000 events of each control.
- Use the compensation setup to calculate the spillover of fluorophores and apply it to the experiment's cytometer settings.
- Recording sample data
- Display FSC and SSC in the acquisition software and set a gate around leukocytes.
- Based on the leukocyte gate, display dot plot SSC/CD45 and gate for CD45+ cells to separate from conidia.
- Display CD45+ cells in a dot plot CD14/CD66b and gate monocytes (CD14+) and neutrophils (CD66b+) separately.
- Display neutrophils in a dot plot anti-FITC/FITC.
- 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.
- Repeat steps 3.5.4 and 3.5.5 for the monocyte gate.
- 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 |
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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 |