Home › Immunology › Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus
Immunology JoVE (Open Access) Citable · DOI

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

DOI: 10.3791/55444-v
What you'll learn
  • ✓ Culture and fluorescently label A. fumigatus conidia for live imaging
  • ✓ Isolate primary human neutrophils and monocytes from blood
  • ✓ Quantify antifungal activity and phagocytosis in real-time using video microscopy
  • ✓ Analyze host–pathogen interactions via flow cytometry and live-cell imaging
Protocol

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

Difficulty
advanced
Total time
~3–4 days (culture preparation 1–2 days; cell isolation ~4 hours; live imaging ~2–3 hours per experiment)
Model organism
Human primary neutrophils and monocytes; Aspergillus fumigatus
Biosafety
BSL-2

Steps

1
Culture and label Aspergillus fumigatus conidia

Grow A. fumigatus and prepare fluorescent reporter conidia for use as imaging substrate. This labeling allows real-time tracking of fungal viability and phagocytosis.

▶ 00:54
2
Isolate human monocytes from peripheral blood

Extract and purify monocytes from donor blood using cell separation techniques. Isolated cells will be used for immune response assays.

▶ 04:28
3
Isolate human neutrophils from peripheral blood

Separate and prepare primary neutrophils from blood samples. These cells are key effectors of antifungal immunity.

▶ 07:30
4
Perform live-cell video microscopy of immune interactions

Co-culture primary immune cells with fluorescent A. fumigatus conidia and capture real-time imaging data on fungicidal activity, phagocytosis, and cell migration.

▶ 08:30
5
Evaluate and interpret antifungal immune response data

Analyze live-imaging and flow cytometry results to quantify fungal killing, phagocytosis rates, and inhibition of fungal growth by primary immune cells.

▶ 09:31
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