Vyb flow cytometer

Tau bioactivity was measured as described [21 (link)], using human embryonic kidney (HEK) cells expressing a CFP/YFP FRET biosensor containing the tau repeat domain (ATCC, cat no. CRL-3275). Briefly, cells were cultured in 96-well plates to 60% confluency. Lysates were mixed with 1% lipofectamine 2000 in OPTI-MEM and 1 ug of total protein was added per well. After incubation for 14–18 h, cells were rinsed in PBS, trypsinized, and fixed with 4% paraformaldehyde. A Miltenyi VYB flow cytometer was used to measure mean FRET intensity and the percentage of FRET-positive cells per well. Multiplication of these values yielded the integrated FRET density (IFD). In addition, an AD positive control sample and a no pathology negative control sample were run on each plate and used to normalize values for comparisons across all samples. All samples were prepared in triplicate.

Biosensor cell lines were harvested with 0.05% trypsin, and quenched with media (DMEM + 50% FBS, 1% Pen/Strep, 1% Glutamax). Cells were centrifuged at 500 x g and resuspended in 4% PFA in 1x PBS. Cells were subsequently centrifuged at 500 x g, resuspended in flow buffer (HBSS + 1% FBS + 1 mM EDTA), and stored for less than 24 hours prior to performing flow cytometry. All flow cytometry for biosensor cells treated with mouse and human-derived tissue was performed using a Miltenyi VYB flow cytometer. Flow cytometry data was analyzed as previously described [10 ]. Integrated FRET density was calculated as integrated FRET density (IFD) = (percentage of FRET-positive cells)*(median fluorescence intensity). IFD was normalized to negative control samples.