Real-Time Assessment of the Antifungal Activity of Primary Human Immune Cells
JoVE Encyclopedia of Experiments
Immunology
0 views • 2:22 min • July 8th, 2025
Begin with a glass-based imaging dish containing a suspension of human neutrophils — or immune cells and visualize it under a fluorescence imaging system.
Introduce genetically modified fluorescent Aspergillus reporter conidia — or fungal spores, and capture images at regular intervals up to 6 hours.
The fungal spores express red fluorescent proteins and are externally labeled with non-degradable magenta fluorophores — helping to distinguish dead cells from live ones.
During incubation, neutrophils interact with pathogen-associated molecules on conidia through pattern recognition receptors, facilitating their engulfment within a phagosome.
Later, the phagosome fuses with the enzyme-containing lysosome, forming a phagolysosome.
Inside the phagolysosome, the reactive oxygen species and enzymes degrade conidia.
During live cell imaging, at the initial time point, intense red and magenta fluorescence within the phagosome of neutrophils indicates the presence of live conidia.
Over time, a decrease in red fluorescence with the retention of magenta fluorescence indicates the degradation of conidia, confirming the antifungal activity of neutrophils.
To visualize DsRed and AlexaFlour 633 during microscopy, preheat a microscope heater and turn on the microscope and the computer. Next, add 100 microliters of 3 times 10 to the 6th per milliliter FLARE conidial suspension to the appropriate wells of the imaging dish. Record the time that FLARE conidia are added.
Mount the imaging dish on the microscope stage. Then, in the Acquisition settings, set the Laser Power to 10% and Exposure Time to one second, and adjust the camera sensitivity for a clear but not overexposed image of DsRed and AF 633, and set up a list of stage points. Initialize imaging when all points are in focus, the fluorescent channels are optimized, and the cycle time and duration are set.
Overview
This study investigates the interaction between human neutrophils and genetically modified fluorescent Aspergillus reporter conidia using live cell imaging. The focus is on visualizing the antifungal activity of neutrophils over time.
Key Study Components
Area of Science
- Immunology
- Cell Biology
- Microbiology
Background
- Neutrophils are key immune cells involved in combating fungal infections.
- Fluorescent labeling helps differentiate between live and dead cells.
- Understanding neutrophil interactions with pathogens is crucial for developing antifungal therapies.
- Live cell imaging allows for real-time observation of cellular processes.
Purpose of Study
- To visualize the dynamics of neutrophil interactions with Aspergillus conidia.
- To assess the antifungal activity of neutrophils through fluorescence imaging.
- To understand the mechanisms of phagocytosis and degradation of fungal spores.
Methods Used
- Use of a glass-based imaging dish for cell suspension.
- Introduction of genetically modified fluorescent Aspergillus conidia.
- Live cell imaging over a period of 6 hours.
- Fluorescence microscopy to monitor changes in fluorescence intensity.
Main Results
- Initial imaging shows intense red and magenta fluorescence indicating live conidia.
- Over time, a decrease in red fluorescence indicates degradation of conidia.
- Retention of magenta fluorescence confirms the presence of dead cells.
- Results demonstrate the antifungal activity of neutrophils through phagocytosis.
Conclusions
- Neutrophils effectively degrade Aspergillus conidia as shown by fluorescence changes.
- Live cell imaging is a powerful tool for studying immune responses.
- The study enhances understanding of neutrophil functions in antifungal immunity.
Frequently Asked Questions
Executive Industry Relevance
This method enables real-time visualization of antifungal immune responses using primary human neutrophils and fluorescent fungal reporters, providing a physiologically relevant system for early-stage target validation in antifungal drug discovery. By quantifying pathogen degradation kinetics through dual-color fluorescence tracking, it supports mechanistic de-risking of immunomodulatory candidates and enhances predictive confidence in lead selection. The assay bridges discovery biology and preclinical evaluation by delivering quantitative, reproducible readouts of innate immune function against clinically relevant pathogens.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Interrogates therapeutic hypotheses by directly measuring neutrophil-mediated fungal killing as a functional readout of immune activity.
- Operational Value: Uses primary human immune cells to de-risk target engagement and mechanism of action in a disease-relevant system.
- Predictive Value: Enables assessment of compound effects on phagolysosomal degradation kinetics, supporting lead identification and portfolio triage.
Screening & Assay Development
- Assay Readiness: Generates standardized, quantitative fluorescence outputs (red/magenta intensity ratios) suitable for high-content screening platforms.
- Reproducibility: Dual-fluorophore design distinguishes live from dead fungal elements, reducing false positives and enabling reliable compound evaluation.
- Scalability: Compatible with glass-based imaging dishes and automated microscopy, supporting assay miniaturization and multi-well formats.
Translational & Preclinical Research
- Disease Relevance: Models human neutrophil response to Aspergillus fumigatus, a clinically significant pathogen in immunocompromised patients.
- Translational Continuity: Connects innate immune mechanism discovery to preclinical efficacy testing through conserved phagolysosomal pathways.
- Risk-Adjusted Decisions: Supports go/no-go criteria based on antifungal activity thresholds derived from immune cell-pathogen interaction dynamics.
Pipeline & Workflow Integration
The assay fits within the discovery continuum from target validation through lead optimization to preclinical immune profiling, particularly for immunomodulators and host-directed antifungals.
- Discovery Biology: Supports hypothesis testing of immune modulators by quantifying real-time pathogen clearance in primary human neutrophils.
- Screening: Delivers assay-ready, reproducible fluorescence readouts that enable comparison of test compounds against pathogen challenge.
- Analytics: Provides time-resolved quantitative measurements of conidial degradation, facilitating mechanistic interpretation of immune activation or suppression.
- Translational Research: Aligns with preclinical validation by using human cells and clinically relevant fungal strains to predict in vivo immune responses.
- Enterprise Reuse: Establishes a reusable platform for antifungal immune profiling across multiple projects and target classes.
Operational & Enterprise Impact
- Scientific Value: Delivers predictive confidence in antifungal mechanisms by visualizing phagolysosomal function in primary human immune cells.
- Operational Value: Ensures standardization and reproducibility through dual-fluorophore discrimination and defined imaging parameters.
- Strategic Value: Improves go/no-go decisions by reducing mechanistic ambiguity in immune-mediated antifungal activity.
- Portfolio Impact: Enables risk-adjusted prioritization of candidates based on quantitative antifungal activity profiles.
Implementation Considerations
- Requires expertise in primary human immune cell isolation, culture, and fluorescence microscopy.
- Dependent on access to genetically modified fluorescent Aspergillus conidia and compatible imaging infrastructure.
- Necessitates standardization of cell-to-pathogen ratios, incubation times, and imaging intervals across users and sites.
- Adaptation to other immune cells (e.g., monocytes) or pathogens may require optimization of phagocytosis and degradation kinetics.
- Practical limitations include donor variability in neutrophil function and the 6-hour imaging window constraining long-term activity assessment.
Biopharma FAQs
Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry
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Last updated: 29 August 2026