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