V plex msd

Fecal solute preparations were adapted from previously described methods with significant alterations (21 (link), 22 (link)). Specifically, an aliquot of two grams of frozen feces was obtained and mixed with 8 mL of saline solution (DPBS, Protease Inhibitor, EDTA, DNase). Samples were homogenized for 1 min and placed on ice for 30 min. Samples were then ultra-centrifuged at 12,000 rpm for 45 min at 5°C, and the supernatant was passed through a 0.2 μm filter, aliquoted and stored at -80°C until testing. Total protein levels were assessed using a Bicinchoninic acid (BCA) assay. Standard sandwich ELISAs were used to measure fecal calprotectin (Epitope Diagnostics, San Diego, CA), IL-22 (eBioscience/Thermofisher, Waltham, MA), sCD14 (Hyclone, Uden, Netherlands) and sIgA (BioVendor, Brno, Czech Republic), while multi-plex ELISAs (MesoScale Diagnostics, Rockville, MD) were used to measure multiple analytes simultaneously each following manufacturer’s protocols. The following V-Plex MSD (MesoScale Diagnostics, Rockville, MD) kits were utilized: Proinflammatory Panel 1 (IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, TNF-α), Vascular Injury Panel 2 (SAA, CRP, VCAM-1, ICAM-1), and Cytokine Panel 1 (GM-CSF, IL-1α, IL-5, IL-7, IL-12/23p40, IL-15, IL-16, IL-17A, TNF-β, VEGF-A).

Inflammatory proteins were measured on the V-Plex MSD electrochemiluminescence multi-spot assay platform (MesoScale Diagnostics, Rockville USA).100 mg of fresh frozen grey matter from AD cases (n = 67) was homogenised at a tissue concentration of 20% w/v in RIPA lysis buffer (Thermo Fisher Scientific) by use of a handheld homogeniser (Thermo Fisher Scientific); the buffer was supplemented with protease inhibitors (Complete Mini, Sigma Aldrich) and phosphatase inhibitors (Thermo Fisher Scientific). Total protein concentration in the supernatant was measured by BCA Protein Assay Kit (Thermo Fisher Scientific). 12.5μl of brain homogenate (1:4 dilution) was used for each assay according to the manufacturer’s protocol. The following V-PLEX human biomarker 40-PLEX kits were used: pro-inflammatory panel 1, cytokine panel 1 and vascular injury panel 2. Each plate was imaged on the Meso QuickplexSQ120 (MesoScale Discovery) according to manufacturers’ instructions for 384-well plates to obtain absolute protein levels (pg/ml). Frozen blocks from 4 controls and 2 multiple sclerosis brains containing chronic inactive, acute and chronic active lesions were used as negative and positive controls, respectively.