A Cell-Based Assay to Assess the Tau Protein Uptake by Microglia

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.

On the first day of the experiment prepare the BV-2 cells by first washing them with 1x PBS in the flask in which they have been cultured. Then, incubate them with 0.05% trypsin EDTA at 37 degrees Celsius and 5% CO₂ until they detach from the flask. Resuspend the cells in culture medium by pipetting up and down 3 to 5 times.

Count cells and prepare a cell suspension with a final concentration of 100,000 cells per milliliter in culture medium containing 200 micrograms per milliliter heparin. Add 250 microliters of this cell suspension per well in a 96-well tissue culture flat-bottom plate. Incubate the plate at 37 degrees Celsius with 5% CO₂ overnight.

After thawing previously prepared pH dye-Tau aggregates on ice, dilute them in 65 microliters per condition of serum-free medium to make a 500-nanomolar solution. Dilute antibodies in 65 microliters of serum-free medium to achieve double the desired concentration. Mix pH dye-Tau aggregates and antibodies in a 96-well U-bottom plate. Seal this dilution plate and incubate overnight at 37 degrees Celsius.

On the following day, remove the medium from the 96-well plate with BV-2 cells. Wash the cells in each well with 100 microliters of room temperature 1x PBS. Remove PBS from the BV-2 cell plate. Use a multichannel pipette to transfer 125 microliters of immunocomplexes from the dilution plate to each well of a 96-well BV-2 cell plate, and incubate for two hours at 37 degrees Celsius with 5% CO₂.

After the incubation, remove the immunocomplexes from the cells and wash the cells in each well with 100 microliters of room temperature 1x PBS. After removing the 1x PBS, add 50 microliters of 0.25% trypsin-EDTA, and incubate for 20 minutes at 37 degrees Celsius with 5% CO₂. After that, add 200 microliters of culturing medium and resuspend the well by pipetting up and down to detach the cells.

Transfer the cells to a 96-well U-bottom plate and centrifuge it at 400 times g for 5 minutes at 4 degrees Celsius. Place the plate on ice and remove the medium. Add 150 microliters of ice-cold 1x PBS, and centrifuge again at 400 g for 5 minutes at 4 degrees Celsius. Place the plate on ice and wash again by removing previously added 1x PBS and adding 150 microliters of ice-cold PBS per well. Centrifuge again under the same conditions.

Place the plate on ice and wash the cells by removing the previously added PBS, and adding 150 microliters FACS buffer per well. Centrifuge again at 400 g for 5 minutes at 4 degrees Celsius. Place the plate on ice, and remove the previously added FACS buffer, and resuspend the cells in 200 microliters FACS buffer per well. Immediately proceed to analyze by FACS to acquire 20,000 events in the live-cell gate.