Microglial Phagocytosis Assay: An In Vitro Assay to Establish Microglial Phagocytosis Model for Neuroblastoma Cells Using iPSC Macrophages

Microglia are brain macrophages that help remove pathogens, cell debris, and cancerous nerve cells or neuroblastoma by a process called phagocytosis.

To mimic phagocytosis in vitro, begin with a culture of neuroblastoma cells fixed in paraformaldehyde.

Paraformaldehyde, a chemical fixative, causes random distribution of membrane phospholipids like phosphatidylserine, resulting in their translocation to the cell membrane's outer surface while simultaneously stabilizing the cell structure.

Now, add a pH-sensitive amine-reactive fluorescent dye to the neuroblastoma cell culture and incubate.

The dye molecules bind to the amine groups of phosphatidylserine on the neuroblastoma cell surface.

Centrifuge the tube and remove the supernatant containing unbound dye molecules. Rinse the cell pellet with a suitable buffer and centrifuge.

Remove the supernatant and resuspend the pellet in the culture medium.

Next, seed pre-stained macrophages in a culture plate. Stained macrophages will appear red with a blue nucleus.

Add stained neuroblastoma cells to this culture and incubate.

The macrophage-phagocytic receptors recognize the exposed phosphatidylserine on neuroblastoma cells and internalize them in membrane compartments called phagosomes.

The phagosomes migrate and fuse with lysosomes, exposing neuroblastoma cells to the lysosome's low pH environment. Low pH activates the dye molecules on neuroblastoma cells, causing the dye color to change.

Under a fluorescence microscope, the phagocytosed neuroblastoma cell appears yellow inside the red macrophages.