Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1

The overall goal of this analysis is to quantify phagosome velocity in HIV-1 infected macrophages using a simple manual tracking method. The advantage of this protocol is to provide an easy method to calculate the relative movements of groups of objects within a cell that can be either moving or deformed. This method provides insight into the movement of phagosomes in GFP-positive infected macrophages but it could be used for other intercellular compartments in fluorescently tagged cells.

After infecting human monocyte-derived macrophages and opsonizing sheep-bred blood cells, according to the text protocol, set up a phagocytosis assay using a confocal imaging system, such as a spinning disc microscope, equipped with a heating chamber at 37 degrees Celsius, with carbon dioxide. Prior to the experiment, turn on the heating chamber to heat the microscope stage to 37 degrees Celsius. Then turn on the microscope and computer, and load the imaging software.