Sulfo tag

CSF was collected and stored according to JPND-BIOMARKAPD guidelines [32 (link)]. AD CSF biomarkers (Aβ42, total Tau and phospho Tau₁₈₁) were analyzed as a part of the routine diagnosis (Innotest; Fujirebio, Ghent, Belgium) [33 (link)].
The VEGF levels in CSF were determined using a Meso Scale Discovery (MSD) cytokine-V-PLEX single cytokine assay (cytokine panel1 human), following the manufacturer’s protocol. The kit was validated for the analysis of VEGF in CSF in earlier studies [34 (link), 35 (link)]. Briefly, MSD plates were precoated with capture antibodies on a defined spot. CSF samples were diluted twice using sample dilution buffer and 50 μl of CSF samples were added to each well. The samples were incubated for 2 h while shaking. Plates were washed with 150 μl of washing buffer three times, after which 25 μl of detection antibodies conjugated with electrochemiluminescent labels (MSD SULFO-TAG) were added and were subsequently kept for incubation for 2 h with shaking. Plates were washed again three times using 150 μl of washing buffer and 150 μl reading buffer was added to each well. The MSD buffer added created a chemical environment for electrochemilumiscence. The plates were subsequently analyzed using a MSD imager (Sector Imager 2400) where a high voltage was applied, enabling the captured labels to emit light.

The V-PLEX Proinflammatory Panel 1 Mouse Kit (#K15048D, Meso Scale Discovery = MSD, Rockville, MD) was used to analyze the perilymph, CSF, and blood samples. It is an immunoassay based on electrochemiluminescence that quantifies the levels of the following cytokines: IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, CXCL1, IL-10, IL-12p70, and TNF-α. Briefly, samples and corresponding standards (calibrators) were added into wells of MSD MULTI-SPOT 96-well plates, pre-coated with capture antibodies, and incubated for 2 h at room temperature. Plates were washed three times with 150 μL/well of washing buffer, and 25 μL of detection antibody solution containing antibody conjugated with electrochemiluminescent labels (MSD SULFO-TAG) was applied into each well. Samples were incubated for an additional two h, then washed as described before, and treated with 2X Read Buffer T. Electrochemiluminescence was measured on the matching MSD SI2400 instrument. Concentration of cytokines was determined from electrochemiluminescence signals fitted to the calibration curve.