Quickplex q120 reader

Meso Scale Discovery (MSD) kits were used to measure the levels of Aβ42, p-Tau, and t-tau in the RIPA fraction of the human brain. Serial dilutions of the RIPA fractions were tested to measure protein levels accurately. Before performing the assay, protein levels were measured using a Thermofisher BCA Assay Kit. With those results, 1 μg of protein from the soluble fraction was diluted to evaluate Aβ42, t-tau and p-Tau levels. The plates were developed and read using a QuickPlex Q120 reader (Meso Scale Diagnostics). ELISA plates from Abcam (ab269555) were used to measure the levels of Gal-3 (detection range 58.8–2000 pg/ml) in tissue homogenates. The protocol was carried out according to the manufacturer’s instructions. A Biotek Synergy 2 was used to read the ELISA Gal-3 assay.

Secreted Aβ₄₀ and Aβ₄₂ levels were measured using Meso Scale Discovery. Mouse primary brain cultures from WT or APP/PS1 incubated for 24 h with control (fresh neurobasal media), ApoE3 KI or ApoE4 KI astrocyte-conditioned media were collected. Aβ levels in media were assessed with the 4G8 kit (#K15199E) following the manufacturer's protocol. Plates were read using a QuickPlex Q120 reader (Meso Scale Diagnostics). Total protein in cell lysates was determined by BCA assay and used to normalize the Aβ levels to the concentration of total proteins.

A Mesoscale Discovery platform (MSD, from Rockville, Maryland, USA) was used to evaluate the cytokine levels by a proinflammatory reading plate for IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12 and TNF-α. To measure the cytokines in mouse brain soluble fractions, we pulled together 50 μg of the S1 and S2 fraction. Moreover, MSD plates were used to test the levels of human Aβ₄₀ and Aβ₄₂ in the soluble fraction of the brain extraction from WT and 5xFAD at different ages, 2, 6 and 10 weeks. Serial dilution of the soluble fraction was tested in order to obtain an accurate measure of the protein levels. The plates were developed using the 4× reading buffer diluted 1 time with distilled water and the plates were read using the QuickPlex Q120 reader from Mesoscale. The detection range of the different cytokines presented is the next (IL1β 1670-0,408 pg/mL: IL4, 1660-0,405 pg/mL; IL12, 32200-7,86; IL10, 3410-0,833). Other cytokines evaluated not included but not included in the results: IFN-γ (938-0,229 pg/mL), IL2 (2630-0,642 pg/mL), IL5 (967-0,236 pg/mL), IL6 (5720-1,40), KC/GRO (1980-0,483 pg/mL), TNF-α (627-0,153). Aβ₄₀ detection range (15100-3,69 pg/mL), Aβ₄₂ detection range (2280-0.557 pg/mL).