This study investigates the process of astrocyte-mediated phagocytosis of synaptosomes, which are neuronal fractions rich in synaptic vesicles. The methodology involves using a pH-indicator conjugated synaptosome suspension to visualize the engulfment process by astrocytes in vitro.
Synaptosomes are isolated neuronal fractions rich in synaptic vesicles. These synaptosomes are engulfed by astrocytes - a population of immune cells in the brain - by a process known as phagocytosis.
To study astrocyte-mediated phagocytosis in vitro, begin by taking a culture plate containing a monolayer of astrocytes. Now, add pH-indicator conjugated synaptosome suspension to the plate. This indicator dye has the property to fluoresce exclusively at a low pH. Incubate the plate for the desired duration.
During incubation, phosphatidylserine or PS present on the outer membrane of the synaptosome allows it to bind to the corresponding PS receptors on the astrocyte membrane. This facilitates the settlement of synaptosomes at the base. Aspirate the spent media along with unbound synaptosomes from the plate.
Next, supplement the plate with phagocytosis-promoting factors and incubate. Subsequently, image the plate using a fluorescence microscope. Upon binding to surface, astrocytes engulf the synaptosomes causing them to enter the phagosomes. Once inside the phagosomes, the low pH causes the indicator dye molecules to fluoresce brightly.
Real-time imaging reveals the presence of bright fluorescent spots corresponding to the pH indicator conjugated synaptosomes engulfed by the astrocytes. Eventually, these spots disappear, indicating successful degradation of synaptosomes.
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 1 milliliter of DPBS per wash. After the last wash, add 300 microliters of immunopanned astrocyte basic medium and 5 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 phosphatidylserine receptors on the astrocytes at 37 degrees Celsius and 5% carbon dioxide. After 40 minutes, discard the supernatants and quickly but gently wash the wells three times with 1 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.
繁體中文
