Ficoll separation

A specimen from the meninges and at least one specimen from peripheral blood or spleen were collected from each individual at autopsy. Whole blood was first centrifuged to remove the plasma, after which peripheral blood mononuclear cells (PBMC) were isolated by Ficoll separation (Sigma Millipore). Splenic and meningeal tissues were processed into single-cell suspensions, prior to cell sorting. CD3⁺ T cells and CD14⁺ macrophage lineage cells were isolated by sequential positive selection (CD3⁺ then CD14⁺) from PBMC and splenic or meningeal single-cell suspensions using magnetic activated cell sorting (MACS), per the manufacturer’s protocol (Miltenyi). The estimated number of cells (cell genome equivalents [CGE]) was calculated based on the amount of DNA used for amplifications using the ratio of 6 pg DNA/cell (50 (link), 51 (link)).

Tibias and femurs from C57/KaLwRij mice were flushed, the cells subjected to ficoll separation (Sigma-Aldrich) and mononuclear cells were cultured for 3 days in αMEM/10% FBS/1% P/S supplemented with 100 ng/ml recombinant murine M-CSF (Prepotech, London, UK). The monocytes were re-seeded on day 4 at a density of 6500 cells/cm² and 100 ng/ml recombinant murine sRANKL (Prepotech) was added to the culture medium to induce osteoclast differentiation. The medium was refreshed on day 7 and cultures were stopped on day 10. RAW264.7 cells were seeded at a density of 30,000 cells/cm² in αMEM/10% FBS/2 mM L-glutamine/1% P/S and 30 ng/ml recombinant murine sRANKL (Prepotech, London, UK). On day 3, the differentiation medium was refreshed and on day 4 the cultures were stopped. At the end of osteoclast cultures, cells were fixed in 4% paraformaldehyde, lysed for RNA extraction or stained for TRAP activity using the Leukocyte Tartrate-Resistant Acid Phosphatase kit (Sigma-Aldrich) according to the supplier's protocol. Actin ring formation was assessed by staining cultures with phalloidin-FITC (Sigma-Aldrich) followed by analysis on a Nikon A1R confocal fluorescent microscope (Nikon Instruments Europe, Amsterdam, the Netherlands)