Encyclopedia of Experiments: Immunology
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Transcript
Begin with a flat-bottom multi-well plate containing adhered murine microglia — phagocytic cells in the brain.
Introduce a serum-free medium containing immunocomplexes. These complexes consist of antibodies attached to phosphorylated Tau protein aggregates, which are formed during certain neurodegenerative diseases.
The Tau aggregates are labeled with a pH-sensitive green fluorescent dye.
During incubation, these immunocomplexes interact with the Fc receptors of microglial cells, facilitating their engulfment.
This immunocomplex-containing endosome fuses with a lysosome, forming an endolysosome. The acidic environment of the endolysosome causes the dye molecules to fluoresce.
Wash to remove the non-internalized immunocomplexes.
Treat with trypsin-EDTA solution to detach the cells.
Transfer the cell suspension to a U-bottom plate positioned over ice to stabilize the endolysosome. Pellet the cells and discard the supernatant.
Wash and resuspend the cells with FACS buffer.
Using FACS, identify the single live cells and quantify the green fluorescence intensity to assess Tau uptake by microglia.
