R plex

Proteins were isolated from retinal or choroid tissue of p17 and p31 Vldlr^−/− and control mice (n = 5 per group) using RIPA buffer (R0278, Sigma-Aldrich) containing protease (Complete Tablets Mini, 0463159001, Roche, Basel, Switzerland) and phosphatase inhibitors (Phosstop, 04906845001, Roche). The amount of isolated protein was measured using a colorimetric assay (Pierce™ BCA Protein Assay Kit, 23225, Thermo Fisher Scientific). To examine cytokine concentrations in retinal samples, we used a multiplex electrochemiluminescence panel (R-Plex, Meso-Scale Discovery, Rockville, MD, USA) according to the manufacturer’s instructions. This kit simultaneously measured the protein levels of CC-chemokine ligand (CCL12) and Vascular Endothelial Growth Factor A (VEGFA) in the same samples. These factors were chosen based on the RNA Seq results of this study and represented relevant or differentially expressed genes in RAP microglia compared to steady-state microglia. For statistical analysis, all values below the detection limit were assigned to half of the respective values of the detection limit.

Serum neurofilament light chain (NfL) was evaluated as a biomarker of neurodegeneration (F217X, R-PLEX, Meso Scale Diagnostics, LLC, range: 5.5–50,000 pg/ml, all samples were run in duplicate and data were within the range of the assay)^{85 (link),86 (link)}.

Myokines were measured in serum samples collected from the participants at baseline and follow-up of the exercise intervention, as well as in the cell-culture supernatants of myotubes derived from biopsy samples obtained at baseline and follow-up timepoints. Myokine quantifications were performed using a single-plex assay (R-plex; SPARC) and a multiplex assay (U-plex; BDNF, FGF21, IL-6, IL-8, IL-10, IL-13, IL-15, and IL-18) from Meso Scale Discovery (Rockville, MD, USA) following the manufacturer’s instructions and as previously published [68 (link),69 (link)]. All antibodies with the exception of SPARC were validated for target specificity. More information about specificity tests can be found in the datasheet of the U-plex kits (https://www.mesoscale.com/ (accessed on 21 March 2024)).