In Vitro Phagocytosis Assay: A Live Imaging Based Method to Study Real-time Astrocyte Mediated Synaptosome Phagocytosis
Transcript
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.
