TIRF Microscopy-Based Visualization of Phagosome Formation and Closure

Published: March 29, 2024

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, 1…

Representative 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 non-covalently fixed on poly-lysine coated coverslips. Images are recorded in TIRF mode to detect the site of phagosome closure and …

Divulgations

The authors have nothing to disclose.

Materials

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 (10×500 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

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TIRF Microscopy-Based Visualization of Phagosome Formation and Closure. J. Vis. Exp. (Pending Publication), e22014, doi: (2024).

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