Live-cell Phagocytosis Assay of Microglia-Like Cells Using Human Synaptosomes

Take a multi-well plate containing microglia-like cells exhibiting phagocytic activity.

Incubate with a nuclear stain to label nuclei.

Add an actin polymerization inhibitor to the negative control well.

During incubation, the inhibitor binds to actin filaments, inhibiting actin polymerization and phagocytosis.

Maintain the plate at low temperatures and add pH-sensitive dye-labeled human synaptosomes--isolated fragments of neural synaptic terminals.

Centrifuge the plate at low temperatures to promote cell-synaptosome interactions but prevent premature uptake.

Transfer the plate to a live-cell imaging system.

In the test well, synaptosomes are phagocytosed and fuse with lysosomes forming phagolysosomes. The lysosomal acidic environment activates the dye, resulting in its fluorescence.

Over time, in the test well, more synaptosomes are engulfed causing a further increase in fluorescence.

However, in the negative control well, actin polymerization is inhibited, preventing synaptosome uptake. The resulting minimal fluorescence confirms the fluorescence dependence on actin-mediated phagocytosis.