The overall goal of the following experiment is to capture the real time dynamic process of phagosome maturation and phagolysosome biogenesis in phagocytes. This is achieved by loading the lysosomal compartments of the cells with fluorescent conjugated dex strand to enable the visualization of lysosomal compartments and transfer of its luminal cargo to phagosomes. As a second step, microparticles are added, which act as a phagocytic model.
Next, the delivery of lysosomal material to the phagosomes is analyzed using live sale imaging and subsequent digital image processing. In order to quantify the phagosome maturation process, the results show the effect of various factors on the process of phagosome maturation based on the delivery of lysosomal content to the phagosomes. The main advantage of this technique of existing methods, such as the evaluation of macrophage maturation in fixed cells is the quantitative readout with a high temporal resolution Begin by dissolving Texas red labeled 70 kilodalton DExT strand or Dex 70 KD in PBS at 20 milligrams per milliliter and store aliquots at minus 20 degrees Celsius.
Next, dilute the Dex 70 KD stock in complete cell culture Docos, modified eagles medium, or DMEM to approximately one milligram per milliliter. Sonicate the DExT strand aliquot in an ultrasonic water bath for five minutes. Then centrifuge the solution in a micro centrifuge at maximum speed for five minutes to remove any clumps or contaminants from the solution.
Carefully move the SuperAgent to a fresh tube while avoiding the pellet at the bottom. Then dilute the solution to a final working concentration of 20 micrograms per milliliter in complete cell culture. DMEM and filter.
Sterilize the solution through a 0.22 micrometer syringe mounted filter. Next, see two milliliters of bone marrow derived mouse macrophages to a concentration of five times 10 to the fourth cells per milliliter. In DMEM in an uncoated glass bottom dish, incubate the cells for at least 12 hours at 37 degrees Celsius in a 5%carbon dioxide buffered atmosphere.
To ensure attachment and acclimatization after attachment, aspirate the medium and add two milliliters of pre-war DExT strand DMEM to the glass bottom dish, and then incubate the cells for two to eight hours following incubation. Wash the cells three times with prewarm complete DMEM. Aspirate the medium after the final rinse and add two milliliters of complete DMEM.
Incubate the cells for four to 12 hours. Then rinse the cells with prewarm phenol red free complete DMEM, and add two milliliters of complete filtered phenol red free DMEM at least 30 minutes before the start of imaging. Next, bring an aliquot of 1%IgG coated beet suspension prepared as described in the accompanying text protocol to room temperature.
Sonicate the solution in an ultrasonic water bath for two to three minutes. Then under a class two biosafety cabinet, add one to six microliters of the bead suspension to the cells. Diffuse the dense cloud of beads by using a pipette to gently mix the suspension in the glass bottom dish.
Then bring the sample to the microscope and focus on the region of interest. Prior to time-lapse imaging. Optimize the settings including scanning, speed magnification, and resolution in order to be able to capture one frame every seven to 20 seconds.
Adjust the excitation emission settings based on the imaging system and probes used and optimize the settings to prevent excessive photo bleaching. Then capture the time lapse video and save it in the native file format of your microscope. Avoid exporting the video in compressed formats such as JPG or PNG.
Next, launch Fiji and Image J distribution containing an image processing package with reorganized tool menus and additional native plugins available for free.Download. Open the file in Fiji and select the video that is to be evaluated. Then set the options, filters and measurement parameters as detailed in the accompanying text protocol.
Next, scan through the images noting the frame in which a fully formed nascent bead phagosome is formed and the F acidic cup is closed. Use the Oval selection tool to select a circular region of interest or ROI on the internalized bead. Ensure the selection fits the outer edge of the bead tightly.
Then go to analyze and click measure to execute the measurement. The result will be displayed in a separate window titled results. In the next frame of interest, adjust the position of the ROI in case the bead has moved and repeat the measurement, which will be added to the list in the results window.
Each analyzed frame can be identified based on the value in the label column. After completing the measurement of all relevant frames, copy the values from the int den column to a spreadsheet application. The column int den depicts the intensities of the selected area.
Using these methods, clear images were captured of bone marrow derived macrophages that had been loaded with the DExT strand probes. This image is from time zero, which is just when the cell completed uptake of the IgG coded bead pointed out here. The images shown here were pseudo colored for better visibility and depict phagosome from zero to 85 minutes after uptake.
The measured phagosome, ROI is outlined with the dashed line and the instances of dextran delivery and accumulation in the phagosome are pointed out by the arrows. Graphs were then generated from these images and show a clear temporal trend of signal association with the phagosome over time. This graph represents an average of 10 phagosomes from the two cells shown here.
While the absolute signal intensity often varies between each analyzed phagosome, the temporal trend is normally very similar. Delivery of Dex 70 KD to the Phagosomes, a plateau was reached within 35 to 40 minutes after phagocytosis. After watching this video, you should have a good understanding of how to evaluate phagosome maturation using time-lapse confocal microscopy.
