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Microscopy & Imaging JoVE (Open Access) Citable · DOI

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

DOI: 10.3791/57566-v
What you'll learn
  • ✓ Isolate resident peritoneal macrophages and human red blood cells from mouse tissue
  • ✓ Label macrophage plasma membrane and opsonize red blood cells for imaging
  • ✓ Acquire and interpret 3D time-lapse confocal microscopy data of phagocytic events
Protocol

Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic cells.

Difficulty
advanced
Total time
~4–6 hours per experiment (including cell isolation, labeling, and imaging acquisition)
Model organism
Mouse (peritoneal macrophages); Human red blood cells (in vitro)
Biosafety
BSL-1

Steps

1
Isolate resident peritoneal macrophages and human RBCs

Harvest resident peritoneal macrophages from mouse peritoneal cavity and isolate human red blood cells. Prepare single-cell suspensions for downstream labeling and imaging.

▶ 01:00
2
Label macrophage membrane and opsonize target cells

Apply fluorescent plasma membrane dye to macrophages and incubate human red blood cells with mouse IgG to enable antibody-mediated recognition. Prepare cells for microscopy imaging.

▶ 04:00
3
Acquire 3D time-lapse spinning disk confocal images

Use spinning disk confocal microscopy to capture single phagocytic events in real time with three-dimensional spatial resolution. Record z-stack image sequences throughout the phagocytic process.

▶ 06:32
4
Analyze and interpret phagocytic imaging data

Review representative time-lapse 3D reconstructions to visualize macrophage membrane dynamics and engulfment of opsonized targets. Evaluate phagocytic kinetics and morphology.

▶ 07:02
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