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.
Optimize the imaging settings, such as scanning speed, magnification, resolution, et cetera, to have at least one cell per field, and to image one frame every minute between 60 to 120 minutes. Place the imaging dish on the microscope stage. Adjust the focus and the location to find only one whole HIV-1 infected macrophage in the field.
Use appropriate excitation and emission settings based on the used imagine system and probe. Include a bright field channel. Optimize its appearance by adjusting the exposure time.
Next, remove the imaging dish and add one milliliter of sheep-bred blood cell, or SRBC suspension, at seven times tenth of the sixth SRBCs per milliliter, to the dish. Centrifuge the dish at 500 times g for two minutes at room temperature to synchronize phagocytosis, then record the time at the end of the centrifugation, and return the dish to the stage. Optimize the focus and set the top and bottom of the stack with a step distance of 0.3 micrometers.
Start acquisition of the Z stacks of GFP and BF images throughout the thickness of the cell, and save the time-lapse video in the native file format of the imaging system. To carry out video editing, in the video editing software, click on the drop-down menu Apps and on the tab Review Multidimensional Data. To open the file, click on Select Base File, then on Select Directory.
In the Data Sets box, select the acquisition to analyze and click on View. To represent the infection in a Z projection, in the Wavelengths box, select the 491 nanometer wavelength and click on the Z projection tab with all planes. Next, to analyze a time sequence, in the Wavelengths box select BF wavelength and choose the optimal plane on the Z axis to distinguish external SRBCs, internal SRBCs, and the nucleus.
Save the video montages by clicking on the Selection Xs tab and then on Load Images. Finally, save the loaded images in tif format. After downloading the Image J manual tracking plugin, open the plugin in Image J, then open the image sequence to be analyzed.
Enter the settings, such as time interval, which represents the amount of time between adjacent frames, and the XY calibration, which represents the distance per pixel. To start the tracking, click on Add Track, and click on an SRBC center at the first time when it is internalized. The next frame appears automatically.
For convenience, to see SRBCs on the BF channel, use the brightness and contrast window during the tracking. Continue to click on the SRBC center in all frames to have different positions during the time. Between each SRBC tracking, click End Track and then Add Track to begin a new track.
The tracking number will change in the second column of the results table. Start to track the nucleus to have its position in all frames by clicking on its center as performed previously. Save the data in a spreadsheet, then open it in spreadsheet software, and create a new spreadsheet file.
Transfer into this new file the time, and the X and Y coordinates of the SRBC and the nucleus. Finally, use the spreadsheet software to calculate the traveled distance of phagosomes containing SRBCs towards the nucleus, and the velocity of the phagosomes during the first fives minutes after internalization of SRBCs according to the text protocol. Analysis of phagosome movement in living HIV-infected macrophages in real time using spinning disc confocal microscopy, is shown here.
The settings for the bright field channel are critical to see the nucleus and for discriminating the external from the internalized SRBCs. This graph plots the distance traveled for a phagosome in non-infected HMDMs in the first five minutes after internalization of an SRBC versus time. The velocity of the phagosome is the slope of the linear regression curve shown here.
Finally, in this study, it was observed that the phagosome velocity during the first five minutes after internalization in HIV-infected HMDMs is lower compared to non-infected HMDMs. These methods demonstrated that phagosome movement to the cell center, and therefore phagosome maturation into phagolysosomes, was impaired in HIV-infected macrophages. After its acquisition, a cell can be further processed for correlative electron microscopy or the whole-cell population can be fixed for classical immunofluorescence microscopy and statistical analysis.
Phagocytosis is very sensitive to temperature and therefore the temperature within the heating chamber and the culture dish medium should be maintained at a constant 37 degrees Celsius throughout the procedure. Don't forget that working with pathogens can be hazardous and precautions such as working in a bio-safety room, and wearing personal protection equipment should be taken according to local legislation.
