Manganese 2 chloride mncl2

Levels of OPG were measured in both plasma as well as in EVs. For this, two EV subpopulations were isolated. The isolation was performed as described in previous publications^{30 (link),31 (link)}. In brief, a subset of EVs co-precipitate with low-density lipid particles (LDL) which allows separation. Magnetic beads were therefore added for both subpopulations (nanomag^®-D plain voor LDL and nanomag^®-D PET-OH for TEX). For the sequential isolation of the EV-LDL subpopulation Dextran Sulphate (DS, 0.05%, MP biomedicals) was used in combination with Manganese II Chloride (MnCl2, 0.05 M, Sigma-Aldrich) (EV-TEX). The TEX subpopulation was precipitated with Xtractt buffer (1:4, Cavadis BV). Subsequently, a bio-plex handheld magnet was used. The remaining pellet containing the EV subpopulations was separated from magnetic bead debris with centrifugation, after removal lysis buffer was added to study the OPG levels carried within the EV subpopulations.

Venous blood was collected in EDTA tubes directly before MPI from the peripheral intravenous cannula. Blood tubes were centrifuged 10 min at 1850×g at room temperature (RT) within 30 min after collection. Plasma was aliquoted and directly stored at − 80 °C. Plasma extracellular vesicle subfractions were isolated using a modified protocol based on the publication of Burstein et al.^{17 (link)}. Detailed description of the isolation protocol used can be found in the supplemental materials. In short, a subset of EVs co-precipitated with Low-Density Lipid particles (LDL) while others co-precipitate with High-Density Lipid particles (HDL), which allows separation. In addition, one subfraction is analysed without the LDL and HDL subfractionation and therefore referred to as TEX subfraction. For the sequential isolation of the subfractions Dextran Sulphate (DS) (MP Biomedicals), Manganese (II) Chloride (MnCl2) (Sigma-Aldrich) solutions and Xtractt buffer (1:4) (Cavadis BV) were used (Supplemental Fig. 1).

Acetone was purchased from Caledon Laboratories Ltd. (Georgetown, ON, Canada). Alkylresorcinol standards were ordered from ReseaChem GmbH (Burgdorf, Switzerland). Potassium phosphate buffer (KPi) was purchased from Mallinckrodt (Paris, KY, USA). Fluorescein, Trolox, 2,2′-azobis(2-methylpropionamidine)dihydrochloride (AAPH), super oxide dismutase, manganese(II) chloride (MnCl₂), 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), glutathione reductase (GR), and 2-vinylpyridine (VP) were purchased from Sigma-Aldrich (Oakville, ON, Canada). Ethylenediaminetetraacetic acid (EDTA), sulfosalicylic acid (SA), glutathione (GSH), bovine serum albumin (BSA), nicotinamide adenine dinucleotide (NADH), 2-mercaptoethanol (MeSH), and nicotinamide adenine dinucleotide phosphate (NADPH) were purchased from BioShop (Burlington, ON, Canada). Oxidized glutathione (GSSG) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).