Live Imaging of Phagosome Migration in HIV-1-Infected Human Macrophages

Abstract

Source: Lê-Bury, G., et al. Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1. J. Vis. Exp. (2016)

This video demonstrates live confocal imaging of HIV-1-infected human macrophages to visualize and quantify phagosome migration using GFP fluorescence and bright field tracking, providing insights into how HIV alters intracellular trafficking during Fc receptor–mediated phagocytosis.

Protocol

1. Live Cell Video Microscopy Phagocytosis Assay

1. Use a confocal imaging system such as a spinning disk system equipped with a heating chamber at 37°C with CO₂ passing through a bottle with water for humidification.
2. Turn on the heating chamber prior to the experiment to have the microscope stage at 37 °C before the beginning of the phagocytosis assay. Turn on the microscope and computer, and load the imaging software.
3. Optimize the imaging settings, such as scanning speed, magnification, resolution, etc., to have at least one cell per field and to image one frame every minute between 60 and 120 min.
   NOTE:

Results

Figure 1. Preparation of control and HIV-infected hMDMs for phagocytosis imaging. Monocytes are purified by adhesion (1) and differentiated into macrophages for 11 days with M-CSF (2). Next, hMDMs are infected with NLR4.3 HIV-1Gag-iGFP i) .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Glass Bottom Dishes 35 mm uncoated 1.5	MatTek corporation	P35G-1.5-14-C Case	For acquisition
RPMI 1640 medium, no phenol red (10x 500 ml)	Life technologies	11835-105	Warm in 37 °C water bath before use ; for phagocytosis assay
Sheep red blood cells (SRBCs)	Eurobio	DSGMTN00-0Q	Conserved in Alsever buffer at 4 °C before use
Confocal Spinning Disk Unit CSU-X1M1	Yokogawa
491 nm 50 mW laser	COBOLT CALYPSO