Luminex 200 platform

CSF samples were collected between 8:00 a.m. and 2:00 p.m. without requiring fasting. This window was chosen because CSF Aβ42 levels during these times represent approximately 95–105% of average Aβ42 over time [32 (link)]. CSF was immediately aliquotted after collection and before freezing, and otherwise we used the ADNI biofluid protocols, including the use of 24-gauge Sprotte needles, aspiration syringes, and transfer into 15-ml polypropylene tubes. To avoid measurement bias related to well positions, sample positions were randomized for each biomarker assay.
CSF biomarkers were measured following the manufacturers’ protocols: Aβ42, t-tau, and p-tau₁₈ levels were measured using the INNO-BIA AlzBio3 immunoassay (Fujirebio, Malvern, PA, USA) in a Luminex 200 platform (Luminex, Austin, TX, USA) [33 (link)] (average interplate coefficients of variation of 13% for Aβ42, 10% for t-tau, and 11% for p-tau₁₈₁), Aβ40 levels using the INNOTEST® enzyme-linked immunosorbent assay (ELISA) (Fujirebio), α-synuclein levels using the Novex® ELISA (Thermo Fisher Scientific, Waltham, MA, USA), NfL levels using the NF-light® ELISA (Uman Diagnostics, Umeå, Sweden), and sICAM-1 and sVCAM-1 levels using a commercial multiplex kit (MILLIPLEX® MAP Human Neurodegenerative Magnetic Bead Panel 3 [HNDG3MAG-36 K]; EMD Millipore, Billerica, MA, USA).

Each experimental condition/technical repeat was analyzed in duplicate (i.e., two samples per experimental cell culture well). The supernatants were analysed using the Procartaplex™ 39-PLEX, Human Cytokine/Chemokine 39 (Thermofisher, Waltham, MA) using the manufacturer's instructions as previously described,^{17 (link)} with overnight incubation. As described, the time points 0 (for control wells), 24, 48, and 72 h were analyzed. The plates were analyzed on a Luminex 200 platform (Luminex Corporation, Austin, TX). As per manufacture's recommendation, cytokine concentrations were calculated by reference to an eight-point five-parameter logistical standard curve for each cytokine.

Thyroid function was inferred from analyses of total T4, total T3, and TSH levels in the serum. Twenty microliters of serum were used to assay for TSH with a Milliplex MAP Mouse Pituitary Magnetic Bead Panel (Millipore-Sigma) on a Luminex 200 platform (Luminex; Austin, TX, USA). Twenty-five microliters of serum were used to assay for total T4 and total T3 using the AccuDiag^TM ELISA—T4 kit (Diagnostic Automation; Woodland Hills, CA, USA) and the T3 (Total) ELISA kit (Abnova; Taipei, Taiwan), respectively, according to the manufacturer’s instructions.