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Related Experiment Video

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Quantification of Efferocytosis by Single-cell Fluorescence Microscopy
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Quantitative Live-Cell Fluorescence Microscopy During Phagocytosis.

Stella M Lu1,2,3, Sergio Grinstein2,3, Gregory D Fairn4,5,6

  • 1Department of Biochemistry, University of Toronto, Toronto, ON, Canada, M5S 1A8.

Methods in Molecular Biology (Clifton, N.J.)
|November 6, 2016
PubMed
Summary

This study introduces a novel method using fluorescent probes and confocal microscopy to visualize lipid and protein dynamics during phagocytosis in macrophages. This technique allows for real-time, noninvasive analysis of cellular processes.

Keywords:
Confocal microscopyFluorescent probesMacrophagePhagocytosis

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Area of Science:

  • Cell Biology
  • Immunology
  • Microscopy

Background:

  • Phagocytosis is a critical cellular process for pathogen clearance and tissue homeostasis.
  • Phagosome maturation involves dynamic recruitment of proteins and lipid remodeling.
  • Studying these transient events requires high-resolution imaging techniques.

Purpose of the Study:

  • To develop and validate a noninvasive method for analyzing phagocytosis.
  • To investigate the dynamic distribution and metabolism of lipids and proteins during phagocytosis.
  • To quantify phagocytic activity in macrophage cell lines and primary cells.

Main Methods:

  • Utilizing genetically encoded fluorescent chimeric probes.
  • Employing transient transfection in RAW 264.7 macrophage cell line and primary macrophages.
  • Applying fluorescence confocal microscopy for high spatial and temporal resolution imaging.
  • Analyzing the uptake of immunoglobulin-opsonized particles.

Main Results:

  • Demonstrated the capability of fluorescent probes to track lipid and protein dynamics in real-time.
  • Quantified phagocytic activity using confocal microscopy.
  • Visualized the concerted recruitment of effector proteins and lipid changes during phagosome formation and maturation.

Conclusions:

  • The described method provides a powerful tool for studying phagocytosis.
  • This approach enables noninvasive, high-resolution analysis of cellular dynamics.
  • The findings contribute to a better understanding of the molecular mechanisms underlying phagocytosis.