TIRF Microscopy-Based Visualization of Phagosome Formation and Closure

Abstract

Source: Marie-Anaïs, F., et al. "Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM). J. Vis. Exp. (2016).

This video demonstrates a high-resolution total internal reflection fluorescence (TIRF) microscopy technique for real-time visualization of phagosome formation and closure during macrophage-mediated phagocytosis of IgG-opsonized red blood cells (RBCs) attached to the surface of a glass bottom dish. The macrophages extend pseudopodia around the RBCs and engulf them inside the phagosomes, detaching them from the glass surface.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

Note: The plasmid used Lifeact-mCherry is a kind gift of Dr. Guillaume Montagnac, Institut Curie, Paris.

1. Cells and Transfection

Note: RAW264.7 macrophages are grown to subconfluency in complete medium (RPMI (Roswell Park Memorial Institute) 1640 medium, 10 mM HEPES, 1 mM sodium pyruvate, 50 µM β-mercaptoethanol, 2 mM L-Glutamine and 10% FCS (Fetal Calf Serum)) in a 100 mm plate. They are transfec....

Results

Figure 1. Schematic Representation of the "Phagosome Closure Assay" Analyzed by TIRFM. The phagosome closure assay is performed using macrophages transiently expressing one or two fluorescently tagged protein. Macrophages are deposited on IgG-opsonized SRBCs n.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anti-sheep red blood cells IgG	MP Biomedicals	55806
Bovine Serum Albumin heat shock fraction, pH 7, ≥98%	Sigma	A7906
Cell lifter	Corning	3008
Cuvettes 4mm	Cell project	EP104
DPBS, no calcium, no magnesium	Thermo Fischer Scientific	14190-094	Room temperature
Electrobuffer kit	Cell project	EB110
100mm TC-Treated Cell Culture Dish	Corning	353003
Gene X-cell pulser	Biorad	165-2661
Gentamicin solution	Sigma	G1397
Glass Bottom Dishes 35 mm uncoated 1.5	MatTek corporation	P35G-1.5-14-C Case
iMIC	TILL Photonics		Oil-immersion objective (N 100x, NA1.49.), heating chamber with CO2, a camera single photon detection EMCCD ( Electron Multiplying Charge Coupled Device) and a 1.5X lens
Poly-L-Lysine Solution 0.1%	Sigma	P8920-100ml	Dilution at 0.01% in water
RPMI 1640 medium GLUTAMAX Supplement	Life technologies	61870-010
RPMI 1640 medium, no phenol red (10x500 ml)	Life technologies	11835-105	Warm in 37°C water bath before use
Sheep red blood cells (SRBCs)	Eurobio	DSGMTN00-0Q	Conserved in Alsever buffer at 4°C before use