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

Abstract

Source: Byun, Y. G., Chung, W. S. A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes. J. Vis. Exp. (2018)

In this video, we evaluate the phagocytic capacity of purified astrocytes in vitro by live imaging assay. The assay serves as an effective screening platform to identify chemical compounds that can enhance or inhibit the phagocytic capacity of astrocytes.

Protocol

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-conditi....

Results

Figure 1. Schematic of phagocytosis live-imaging assay.

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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
pHrodo red, succinimidyl ester 	Molecula probes	 P36600	For pH indicator conjugation
Fetal bovine serum (FBS) 	Gibco	16000-044
DMEM	Gibco	11960-044
dPBS	Welgene 	LB001-02
<strong>Phagocytosis live imaging assay</strong>
Juli stage&nbsp;	NanoEntek
Time Series Analyzer V3 plugins&nbsp;	https://imagej.nih.gov/ij/plugins/ time-series.html