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

1. Phagocytosis Live-imaging Assay (Figure 1)

1. Prepare a 24-well plate (or any plate/dish that is prepared for live-imaging) in which astrocytes are confluent (generally, 7 to 10 days of incubation after the purification is ideal for stabilizing the cells).
2. Remove the media in each well and wash the wells with 1 mL of Dulbecco’s phosphate buffer saline (DPBS), three times. To minimize air exposure, wash the wells quickly.
3. Add 300 µL of immunopanned astrocyte basic media (IP-ABM) with 5 µL of pH indicator-conjugated synaptosomes and additional factors such as immunopanned astrocyte-conditioned media (IP-ACM) that can modulate glial cell phagocytosis.
4. Incubate the plate in a CO₂ incubator for 40 min. This step allows the pH indicator-conjugated synaptosomes to settle to the bottom of the plates.
5. Remove the media with unbound pH indicator-conjugated synaptosomes and wash each well with 1 mL of DPBS, three times.
   CAUTION: Do not wash harshly. To minimize air exposure, wash the wells quickly.
6. Add 500 µL of IP-ABM with additional factors to test into the wells.
7. Take the plate to a live-imaging instrument and select positions. Adjust focus, exposure time, brightness, and LED power.
   NOTE: The settings for exposure time, brightness, and LED power are variable depending on fluorescence intensity and the purpose of the experiment. In the live-imaging assay with pH indicator-conjugated synaptosomes, we usually set those values as follows: exposure: ~150–200 ms, brightness: 15, and LED power: 4–6.
8. Set the image format, time interval, and the total number of cycles for live imaging. Set the time interval to 1 or 2 h depending on how many positions are selected. 2-hour intervals are recommended when more than 150 positions are selected for live-imaging.
9. Start the live-imaging experiments.