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 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.
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