The overall goal of this experiment is to detect the real time engulfment and degradation kinetics of glial cells, such as astrocytes and microglia, through in vitro live-imaging of the phagocytosis of pH indicator-conjugated synaptosomes. This method can help answer key questions in the neuroscience field, such as how is glial cell phagocytosis regulated in healthy and diseased brains. The main advantage of this technique is that we can effectively monitor and analyze the real time phagocytic kinetics of glial cells through live imaging of in vitro cultured cells.
This method can also be used to screen for factors and compounds that regulate the engulfment and degradation capacity of glial cells. Begin by centrifuging the thawed synaptosome samples for three to four minutes at 21, 092 G and four degrees Celsius. After aspirating the supernatants, re-suspend the pellets in 200 microliters of 0.1 molar sodium carbonate per tube, adding two microliters of pH indicator to each sample after they have been thoroughly mixed.
After a gentle vortex, protect the solutions from light with aluminum foil covers, and place the samples in a twist shaker with gentle agitation for two hours at room temperature. At the end of the incubation, add one milliliter of Dulbecco's PBS, or DPBS, and collect the synaptosomes by centrifugation, re-suspending the pellet in one milliliter of fresh DPBS per wash. After the last centrifugation, re-suspend the non-functional synaptosome pellet in 200 microliters of DPBS supplemented with 5%DMSO.
For live imaging of the synaptosome engulfment, first remove the supernatant from each well of a confluent astrocyte culture, and quickly wash the cells three times with one milliliter of DPBS per wash. After the last wash, add 300 microliters of immunopanned astrocyte basic medium and five microliters of the pH indicator-conjugated synaptosomes, as well as any additional factors that can modulate glial cell phagocytosis. Allow the synaptosomes to settle to the bottoms of the wells and to attach to phosphatidyl serine receptors on the astrocytes at 37 degrees Celsius and 5%Co2.
After 40 minutes, discard the supernatants and quickly but gently wash the wells three times with one milliliter of DPBS per wash. After the last wash, add 500 microliters of fresh medium supplemented with the factors of interest to the appropriate wells and transfer the plate to a live imaging instrument. Select the imaging positions, adjust the focus, exposure time, brightness, and LED power, and set the image format, time interval, and total number of cycles for live imaging as experimentally appropriate.
Then, begin live imaging the phagocytosis experiments. At the appropriate experimental endpoint, open an imagine analysis program and import the time lapse image sequence. Convert the images to 8-bit grayscale and initiate a background subtraction with a rolling bar radius of 50 pixels.
Next, open the Time Series Analyzer V3 plugin and drag the region of interest into the plugin. In the region of interest manager, click Add. In the Time Series V3 plugin, click Get Total Intensity.
Then, save the results and integrate the data. After their preparation, the synaptosomes express phosphatidyl serine on their outer membranes, suggesting a loss of function, and that can be recognized by phosphatidyl serine receptors on astrocytes and microglia. As the pH indicator-conjugated synaptosomes are phagocytosed they emit bright red fluorescents.
To quantify glial cell phagocytosis, the area of red fluorescence signal, or phagocytic index, can be measured. Although astrocytes are efficient at phagocytosing large numbers of pH indicator-conjugated synaptosomes, microglia are faster at engulfing and degrading the synaptosomes. Interestingly, astrocyte secreted factors, which are contained in astrocyte conditioned medium, are essential for increasing both astrocyte and microglia mediated phagocytosis.
Further, mouse MEGF10 knockout astrocytes demonstrated significantly impaired phagocytic capacity compared to wild type cells. After its development, this technique paved the way for researchers in the field of neuroscience and glial cell biology to explore the mechanisms involved in the modulation of glial cell phagocytosis as a potential method for treating various neurological disorders.
