Luminex xmap multianalyte profiling platform

During the trial, plasma specimens were collected at the baseline, 6-, 12- and 24-month visits, and immediately stored at − 80 ℃. For the current study, archived specimens were requested and shipped from the National Institute of Aging (NIA) Aging Research Biobank to Mayo Clinic for analysis.
The concentrations of protein biomarkers in plasma samples were quantified using commercially available multiplex magnetic bead-based immunoassays (R&D Systems) on the Luminex xMAP multianalyte profiling platform and analyzed on MAGPIX System (Merck Millipore). All assays were performed according to the manufacturer’s protocols. The biomarkers quantified with this method included ADAMTS13, eotaxin, Fas, GDF15, ICAM1, IL6, IL7, IL8, IL10, IL15, MCP1, MDC, MMP1, MMP7, MMP9, MPO, OPN, PAI1, PARC, RAGE, RANTES, SOST, TNFR1, TNFR2, TNFα, and VEGFA. Activin A concentration was determined by a Quantikine ELISA Kit (R&D Systems) according to the manufacturer’s instructions.
Assay performance characteristics are reported in Supplemental Table 10. In cases where a biomarker was below the limit of detection in a sample, a value of half of the lowest measured value for that analyte was assigned.

Proteins were selected based on previous work (Schafer et al., 2020 (link)), and full names and abbreviations are shown in Supplemental Table 2. The concentrations of target proteins were quantified using commercially available multiplex magnetic bead‐based immunoassays (R&D Systems) on the Luminex xMAP multianalyte profiling platform and analyzed on MAGPIX System (Merck Millipore) using 220 μL of plasma. MMP9, MMP2, PARC, RANTES, PAI1 and MPO were analyzed on a standard 6‐plex panel using 20 μL plasma diluted 1:100. MMP1, ICAM1, GDF15, TNFRI, uPAR, and Fas were run on a standard 8‐plex panel using 30 μL plasma diluted 1:2. MMP7, MDC, RAGE, SOST, OPN, and TNFR2 were assessed as part of a standard 8‐plex panel using 30 μL plasma diluted 1:2. Eotaxin, MCP1, PDGF‐AA, PDGF‐AB, and VEGF were assessed as part of a performance 12‐plex assay using 30 μL plasma diluted 1:2. TNFα and IL7 were run on a high‐sensitivity 4‐plex panel using 55 μl plasma diluted 1:2 (100ul assay volume). Activin A concentration was determined by a Quantikine ELISA Kit (R&D Systems) using 55 μl plasma diluted 1:2. All assays were performed according to the manufacturer's protocols. Assay performance characteristics are reported in Supplemental Table 7, and a matrix showing the correlations between the SASP biomarkers is presented in Supplemental Table 8.

The concentrations of CCL2, CCL3, CCL7, CCL8, CCL22, CXCL1, CXCL2, G-CSF, GDF-15, ICAM-1, IGFBP3, IL-1α, IL-1β, IL-7, IL-17, LIX, M-CSF, MMP-3, OPN, TNFSF6, TNFRSF1A, TNF-α, and VEGF in plasma were quantified using commercially available multiplex magnetic bead immunoassays (R&D Systems) based on the Luminex xMAP multianalyte profiling platform and analyzed on a MAGPIX System (Merck Millipore). All assays were performed according to the manufacturer’s protocols. Activin A and TGF-β1 concentration were determined by a Quantikine ELISA Kit (R&D Systems) according to the manufacturer’s instructions. Investigators were blinded as to sample identity and treatment of mice.