Phospho thr231 total tau kit

Quantitative biochemical analyses of human Aβ soluble and insoluble fraction levels were acquired using the V-PLEX Aβ Peptide Panel 1 (6E10) (K15200G-1; Meso Scale Discovery, Rockville, MD, United States). Plates were prepared and read according to the manufacturer’s instructions. Quantitative biochemical analysis of total tau and phosphorylated tau-231 (pTau231) in soluble fractions were also obtained using the Phospho(Thr231)/Total Tau Kit (K15121D-1; Meso Scale Discovery, Rockville, MD, United States). Finally, quantitative biochemical analysis of neurofilament-light chain (NfL) in plasma was performed using the R-Plex Human Neurofilament L Assay (K1517XR-2; Meso Scale Discovery, Rockville, MD, United States).

Culture supernatants were transferred to 15-ml falcon tubes, spun at 2000g and 4°C for 5 min, aliquoted, and stored at −80°C. A sandwich immunoassay (V-PLEX Aβ Peptide Panel 1 kit, #4G8, Meso Scale Discovery) was used in the measurement of β-amyloid species. Manufacturer’s guidelines were followed thoroughly during plate preparation, and samples were diluted 1:1 with Diluent 35 (provided as part of the kit) to avoid matrix saturation. All biological replicates were measured in parallel. Signal readings were performed on a Sector Imager 2400 instrument (Meso Scale Discovery). For assessing sAPPα levels in culture supernatants, we used a sandwich ELISA assay [sAPPα (mouse/rat) (highly sensitive), #27419, Immuno-Biological Laboratories], and samples were diluted 10-fold. Extracellular tau levels were assessed using the Phospho(Thr²³¹)/Total Tau Kit from Meso Scale Discovery following the protocol provided by the manufacturer. Neurofilament-light and Map2 were measured by ELISA using PathScan Total Neurofilament-L Sandwich ELISA kit (#99175, Cell Signaling Technology) and Abcam’s SimpleStep MAP2 ELISA kit (ab253229), respectively.