Multi analyte elisarray

Multi-Analyte ELISArray (Qiagen) was used for the simultaneous detection of different/most common cytokines/chemokines in co-culture supernatants using conventional sandwich-based enzyme-linked immunosorbent assay (ELISA) technique. The 96-well ELISA microplate had been coated with a panel of target-specific capture antibodies, one in each eight-well strip allowing qualitative, relative profiling results from up to six samples. Each kit also included the corresponding detection antibodies, Antigen Standards, and a complete set of reagents. Using this kit, cell culture supernatants were assessed for the presence of cytokines IL-1α, IL-1β IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12α, IL-13, IL-17α, GM-CSF and TNF-1β.

Inflammatory (IL-1β, IL-6, IL-12, CCL2, CCL3, CCL4, IFNγ, IL-17A, and TNFα), anti-inflammatory (IL-4 and IL-10), and inflammation-related cytokine (IL-6 and TGFβ1) measurement was performed on plasma using the sandwich-based enzyme-linked immunosorbent assay (ELISA) with Multi-Analyte ELISArray (Qiagen, Frederick, Maryland, 21704, USA) following the manufacturer’s recommendations (https://www.qiagen.com/us/products/discovery-and-translational research/functional-and-cell-analysis/elisa-assays/multi-analyte-elisarray-kits) (18 ). The optical density was measured at 450nm (OD₄₅₀), and the concentration in pg/mL was calculated based on the logarithmic function y=a ln(x)+b obtained through the standard curves of samples with known concentrations. OD₄₅₀ reads between 0 and 2.5 were considered to be within the linear range for all of the analytes as instructed (https://www.qiagen.com/dk/resources/faq?id=c9815b21-393e-4cef-8968-5d4e67801aa1&lang=en).

The levels of Aβ in Transwell culture medium or tissue supernatants were determined with a sandwich ELISA (Wako, Osaka Japan). For tissues, hippocampi or cortices were homogenised in 4 M guanidine-HCl buffer (pH 8.0) with an ultrasonic homogeniser (Taitec, Saitama, Japan) and incubated at room temperature for 3 h. The homogenates were diluted with 0.1% bovine serum albumin in PBS and centrifuged at 16,000 × g for 20 min. The resulting supernatants were then applied to the ELISA to measure Aβ levels or to Multi-Analyte ELISArray (Qiagen, Hilden, Germany) to measure levels of the proinflammatory cytokines TNF-α, IL-6 and IL-1β according to the manufacturer’s instructions.
EV-associated NCAM-1 and L1CAM were measured by ELISA from MyBiosource (San Diego, CA) and LifeSpan BioSciences (Seatle, WA). Total exosome levels were analysed using PS Capture Exosome ELISA Kit with anti-CD9 antibody (Wako) according to the manufacture’s instruction. For detection of exosomal amyloid fibrils, conformational specific anti-amyloid fibril antibody (mOC87, abcam) and HRP-conjugated anti-rabbit IgG were used as detection antibodies in PS Capture Exosome ELISA Kit.

Mouse cytokine Multi-Analyte ELISArray was carried out according to manufacturer’s protocol (Qiagen). Briefly, Millipore 96-well plates (R & D system) were incubated with mouse sera (1:4) for two hours in room temperature. Detection antibodies were then added and incubated for two hours in room temperature. Avidin-HRP was added for another 1 h under room temperature. Development solution were then added and ELISA reader was used to detect O.D. at 450 nm.

Inflammatory cytokines were determined using a commercial ELISA (Multi-Analyte ELISArray – Qiagen, Valencia, CA, United States). The cytokines were measured by standard ELISA protocol using a panel of 12 inflammatory cytokines: interleukin-1 alpha (IL-1α), interleukin-1 beta (IL-1β), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin-17 alpha (IL-17α), interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF). Cytokines that presented the greatest change in concentration were selected for detailed analysis. These cytokines were analyzed using a standard ELISA protocol (Single Analyte ELISA Kit – Qiagen, Valencia, CA, United States).