at room temperature for 30min to allow each bead to be individually identified and quantified based on fluorescent signals. After an additional round of plate washing, a final volume of wash buffer was added to the plate in place of sheath fluid for the plate to be read and discourage aggregation during the assay. The plate was read using the Luminex® 200 system equipped with xPONENT® 3.1 software. The standard curve and analyte concentrations were calculated using Milliplex Analyst 5.1 software (EMD Millipore Corporation, Billerica, MA, USA). Dilution control procedures identified a prozone effect for total Tau, which is common in Luminex and other ELISA based assays in the presence of high antigen levels (Jacobs, van der Molen, Bossuyt, & Damoiseaux, 2015), and prevented cross-plate standardization. Thus, Median Fluorescence Intensity (MFI) was measured for Tau within a single plate for each brain region analyzed.
