V plex custom human biomarkers kit

CSF samples were obtained by lumbar puncture in the L3/L4 or L4/L5 interspace with patient non-fasting, in the morning. The samples were collected in polypropylene tubes and gently mixed to avoid gradient effects. All samples were centrifuged within 30 minutes at +4 °C at 2000 g for 10 min to remove cells and debris, and then stored in aliquots at −80 °C pending biochemical analysis. The procedure followed the Alzheimer’s Association Flow Chart for CSF biomarkers^{39 (link)}. CSF YKL-40 concentration was measured by solid phase sandwich ELISA according to the manufacturer’s instructions (R&D Systems, Inc., Minneapolis, MN, USA). CRP, SAA, IL-6, IL-8, and MCP-1 were analyzed using V-PLEX Custom Human Biomarkers kit (Meso Scale Discovery, Rockville, MD, USA) according to the manufacturer’s recommendations. α-syn concentration was measured using the Alpha-Synuclein ELISA Kit (Meso Scale Discovery, Rockville, MD, USA). Hb was analyzed using an assay provided by Bethyl lab. Inc. Total tau (T-tau), tau phosphorylated at Thr181 (P-tau), Aβ₄₂ and NfL have previously been described^{40 (link)}.
All analyses were performed using one batch of reagents by board-certified laboratory technicians who were blinded to clinical data. For α-syn, only samples with Hb < 200 ng/L was used.

Cells were plated at 5 × 10⁴ cells per well in 96‐well plates coated with Matrigel. To control the number of cells, the assay was performed relatively soon after seeding. Therefore, the seeding density was higher than that in other assays or usual passages. Three days after plating, culture media were replaced with 100 μL/well of Astrocyte Media containing IL‐1β (Peprotech) or TNFα (Peprotech), harvested 24 h after replacement. Twenty‐five microlitres of conditioned media was subjected to electrochemiluminescence (ECL)‐ELISA analysis by using a V‐PLEX Custom Human Biomarkers kit (IL‐1β, IL‐6, TNF‐α, IL‐4 and GM‐CSF) (Meso Scale Discovery, Rockville, MD) and MESO SECTOR S600 (Meso Scale Discovery). Cytokine concentrations were calculated based on the kit's standard calibration curve using a four‐parameter logistic fit by applying MSD Discovery workbench 4.0 software (Meso Scale Discovery).