Milliplex multiplex assay

Pro-inflammatory cytokines including IL-1β, IL-6, TNF-α, and MCP-1 in serum from all mice were measured in a single batch by Milliplex multiplex assays (EMD Millipore) according to the manufacturer's instructions and using a Luminex 200 multiplex detection system (EMD Millipore).

At the end of the in vivo experiment, blood samples were collected by cardiac puncture and immediately centrifuged at 1200× g for 15 min to obtain the serum. Serum levels of osteoprotegerin (OPG) and a receptor activator of NF-κB ligand (RANKL) were assessed by customized MILLIPLEX^® Multiplex Assays using a Luminex^® instrument (Merck Millipore, Burlington, MA, USA), following the manufacturer’s instructions.

Cytokine levels in plasma and CSF were measured using cytokine protein multiplex assay (MILLIPLEX® Multiplex Assays, Merck KGaA) on a Luminex 200 platform and Bio-Plex Manager 6.2 software (Bio-Rad Laboratories GmbH) according to the manufacturer’s instructions. Plasma and CSF samples (n = 117) of control individuals (n = 21) or patients with AD (n = 18), MCI (n = 7), HD (n = 46), depression (n = 11), FTLD (n = 5) and SCZ (n = 9) were used for Luminex analysis (Supplementary Table 1).

Mandibles with both soft tissue and bone were subjected to bead beating at two 2 min intervals with 2 min of cooling in between using 1.0-mm-diameter zirconia silica beads (BioSpec) in cell extraction buffer (ThermoFisher). The buffer was prepared with a protease inhibitor cocktail (mini cOmplete, Roche) and PMSF protease inhibitor (Abcam) to allow for dissociation and lysis of all soft tissue while leaving the hard tissues intact. MILLIPLEX Multiplex assays (EMD Millipore) were used to probe resulting lysates for IL-6, IL-1β, IL-10 and MCP1 according to manufacturer protocols. Data were acquired on a Luminex 200 system running xPONENT 3.1 software (Luminex) and analysed using a 5-paramater logistic spline-curve fitting method using Milliplex Analyst v5.1 software (Vigene Tech). Data are presented as pg ml⁻¹ normalized to total protein (BCA assay; ThermoFisher Pierce).

The MILLIPLEX multiplex assays (Merck, Darmstadt, Germany) using xMAP technology (Luminex Corporation, Austin, TX, USA) was used to analyse the concentrations of various analytes within the plasma samples. MILLIPLEX magnetic beads panels (Merck) were used to analyse 14 myokines. A full list of analytes can be found in Supplementary Table S1. All procedures were performed according to the manufacturer guidelines. Standard curves were created for each analyte, using standard concentrations depending on manufacturer guidelines for each panel. Two quality controls provided in the kits were added to each panel. Analysis of Luminex panels was performed using the Luminex 200 (Luminex Corporation) instrument; for acquisition, the xPONENT software (v.3.1.7; Luminex Corporation) was used. The median fluorescent intensity was analysed using a 5-parameter logistic curve-fitting to calculate the concentration of analytes in each sample.

The MILLIPLEX multiplex assays (Merck, Germany) using xMAP technology (Luminex Corporation, TX, USA) was used to analyse the levels of various analytes within the plasma samples. MILLIPLEX magnetic beads panels (Merck) were used to analyse 37 analytes across 11 bead panels, including: inflammatory cytokines and chemokines, adipokines, myokines, and other analytes. A full list of analytes can be found in Supplementary Table S1.
All procedures were performed according to the manufacturer guidelines. Standard curves were created for each analyte, using either a six- or seven-standard concentrations depending on manufacturer guidelines for each panel. Two quality controls provided in the kits were added to each panel. Analysis of Luminex panels was performed using the Luminex 200 (Luminex Corporation) instrument, for acquisition the xPONENT software (v.3.1.7; Luminex Corporation) was used, and data analysis was performed using the Masterplex 2010 software (v.2.0.0.68; Mirai Bio Group, CA, USA). The median fluorescent intensity was analysed using a 5-parameter logistic curve-fitting to calculate the concentration of analytes in each sample.