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