Real-Time Assessment of the Antifungal Activity of Primary Human Immune Cells

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.