Home › Cell Biology › Live-cell Video Microscopy of Fungal Pathogen Phagocytosis
Cell Biology JoVE (Open Access) Citable · DOI

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

DOI: 10.3791/50196-v
What you'll learn
  • ✓ Culture and fluorescently label Candida albicans for microscopy
  • ✓ Prepare mouse macrophage cells for live-cell imaging
  • ✓ Perform and record live-cell phagocytosis assay
  • ✓ Analyze stage-specific macrophage-pathogen interactions from video
Protocol

Biopharma Insights We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

Difficulty
intermediate
Total time
~4–5 hours (culture prep, staining, assay setup, and imaging)
Model organism
Mouse J774.1 macrophage cell line; Candida albicans
Biosafety
BSL-2

Steps

1
Culture Candida albicans under specified conditions

Grow C. albicans to appropriate density and physiological state for phagocytosis assay. Ensure yeast cells are viable and uniformly prepared.

▶ 01:44
2
Stain Candida albicans with FITC fluorescent dye

Label C. albicans cells with fluorescein isothiocyanate to enable fluorescence detection during live-cell microscopy imaging.

▶ 02:38
3
Prepare J774.1 mouse macrophage cell culture

Culture and harvest mouse macrophage cells, then plate them at appropriate density on imaging dishes for live-cell assay.

▶ 03:34
4
Set up and execute live-cell phagocytosis assay

Co-incubate fluorescently labeled C. albicans with macrophages and acquire continuous video microscopy data to visualize recognition, engulfment, and intracellular trafficking.

▶ 04:55
5
Analyze stage-specific events from video microscopy

Review and quantify macrophage migration, pathogen recognition, engulfment kinetics, and phagosome maturation stages from recorded videos.

▶ 06:40
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