V plex aβ peptide panel 1 kit

Quantification of Aβ38, Aβ40 and Aβ42 was performed with the V-PLEX Aβ peptide Panel 1 kit (K15200E) and a Quickplex SQ120 instrument (MesoScale Discovery) using 25 µl of cell culture supernatant collected at Day 18 of TNF treatment.

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.

Incubation medium from vehicle-only (control) and pharmacologically treated 3-week-old tissue cultures was collected, frozen immediately with dry ice, and stored at −80°C (vehicle, n = 6 wells with 36 tissue cultures; BACE inhibitor C3, n = 3 wells with 18 tissue cultures; Begacestat, n = 3 wells with 18 tissue cultures). For the detection of Aβ, a V-PLEX Aβ Peptide Panel 1 Kit (MesoScale Discovery, catalog #K15199E, RRID:AB_2827747) was used with a ruthenylated anti-Αβ antibody (4G8 clone). The Aβ peptide concentration was determined using the MSD Discovery Workbench software (MesoScale Discovery).