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Methods Mol Biol. 2018;1784:165-175. doi: 10.1007/978-1-4939-7837-3_16.

Observing Frustrated Phagocytosis and Phagosome Formation and Closure Using Total Internal Reflection Fluorescence Microscopy (TIRFM).

Author information

1
Inserm, U1016, Institut Cochin, Paris, France.
2
CNRS, UMR 8104, Paris, France.
3
Université Paris Descartes, Sorbonne Paris Cité, Paris, France.
4
Inserm, U1016, Institut Cochin, Paris, France. Florence.niedergang@inserm.fr.
5
CNRS, UMR 8104, Paris, France. Florence.niedergang@inserm.fr.
6
Université Paris Descartes, Sorbonne Paris Cité, Paris, France. Florence.niedergang@inserm.fr.
7
Biology of Phagocytes, Department Infection, Immunity, Inflammation, Institut Cochin, Paris, France. Florence.niedergang@inserm.fr.

Abstract

Complementary methods to observe frustrated phagocytosis and phagosome closure using total internal reflection fluorescence microscopy (TIRFM) are described here. Frustrated phagocytosis occurs when phagocytic cells are exposed to an opsonized surface and spread as if trying to engulf it, allowing for the observation of phagocytic spreading and the biochemical events that directly precede it. Phagosome formation and closure is an inherently three-dimensional process though, and cannot be studied in the "frustrated" situation. Here we describe a method to visualize with unprecedented high-resolution phagosome formation and closure in three dimensions. It allows for observation of the base of the phagocytic cup, the extending pseudopods, as well as the precise site of phagosome scission.

KEYWORDS:

Electroporation; Macrophage; Phagocytosis; RAW264.7; Scission; Total internal reflection fluorescence microscopy

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