Bio plex 200 multiplex assay system

Serum cytokine panels quantified levels of IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-15, IL-18, GM-CSF, IFN-γ, IP-10, KC, MCP-1, TNF-α using the CCYTOMAG-90K canine cytokine/chemokine kit from Millipore (Billerica, Massachusetts). Samples were prepared according to the manufacturer’s instructions, with the exception of the following modifications: a) the volume of all reagents used was halved, while keeping the concentration standard after validating this approach by comparison with the recommended volume; and b) the highest concentration of the standard curve was eliminated after the first 4 samples, since no dog’s cytokine/chemokine levels approached this highest value. All samples were analyzed using a Bio-Plex 200 multiplex assay system (Bio-Rad, Hercules, California).

For Aβ40 and Aβ42 detection, V-PLEX Plus Aβ Peptide Panel 1 (6E10) Kit (Meso Scale Discovery (MSD), K15200E] was used. Soluble and insoluble Aβ40 and Aβ42 levels were measured using the PBS and guanidine extracts of the mouse samples following the manufacturer’s instructions, respectively. For Aβ uptake assay, RIPA extracts of BV2 cell lysates were used to measure levels of intracellular Aβ42. Signals were measured on a MESO QuickPlex SQ 120 (multiplexing imager, MSD).
For cytokine and chemokine measurement, PBS-soluble extracts from Abi3^+/+ and Abi3^−/− male mouse cortices were used. Samples were loaded into Bio-Plex Pro Mouse Chemokine Panel (Bio-Rad, 12002231) to measure the levels of 33 chemokines. Cytokine and chemokine levels were determined using the Bio-Rad Bio-Plex 200 multiplex assay system. We were able to detect 25 of them in the brain samples. The concentrations were normalized by total protein levels in the samples.