One of the proposed mechanisms by which therapeutic anti-Tau antibodies reduce pathology in Alzheimer's disease is uptake in clearance of pathological aggregated former Tau by microglia. Here, we present the cell base assay to assess Tau uptake by microglia. This assay represents a useful investigational tool to better characterize the mechanism of action of anti-Tau antibodies.
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 05%trypsin EDTA at 37 degrees Celsius and 5%C02 until they detach from the flask. Re-suspend the cells in culture medium by pipetting up and down three to five 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%C02, 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 multi-channel pipette to transfer 125 microliters of amino complexes 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 amino complexes 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 25%trypsin EDTA and incubate for 20 minutes at 37 degrees Celsius with 5%CO2. After that, add 200 microliters of culturing medium and re-suspend 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 x 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 re-suspend 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. For FACS analysis use the forward scatter area or FSCA versus side scatter area or SSCA density plot to gate on live cells by excluding events with lower forward scatter levels, such as debris and dead cells.
Then within the live cell population use FSCA versus forward scatter height or FSCH to create a singlet gate and exclude cell doublets and aggregates. Then use the events in the singlet gate to generate a pH dye single parameter histogram. Use the generated histogram to first determine the mean fluorescence intensity.
After that determine percentage of pH dye-Tau positive cells by excluding negative cells as determined USING BV-2's only control. CBTau-28.1 antibodies promote an uptake of Tau in BV-2 cells in a dose dependent manner as shown by flow cytometry. The high affinity double mutant CBTau-28.1 antibody mediated Tau uptake in the BV-2 cells to a higher extent than the wild type antibody.
CBTau-28.1 Fab fragments did not increase basal Tau uptake indicating an FC receptor mediated internalization mechanism. Confocal microscopy confirmed antibody mediated increase of Tau uptake. Green pH dye labeled Tau aggregates often co-localized with lysotracker red dye stained acidic organelles thus suggesting presence of Tau aggregates in an endolysosomal compartment.
CBTau-28.1 Fab fragments did not increase Tau uptake again indicating that the uptake was indeed FC mediated. The assay we just described allows us to specifically consistently quantify antibody mediated Tau uptake by microglia. Data generated with this assay can help elucidating the mechanism of action of anti-Tau antibodies thus representing a useful tool to advance anti-Tau antibodies to further step in their development as potential AD treatment.
