Ethylenediaminetetraacetic acid tubes

At each timepoint, 20 ml of peripheral blood was collected in lithium heparin tubes, and blood cell counts were measured using a hematology analyzer (ADVIA 2120i, Siemens, Zurich, Switzerland) (Kim et al., 2012 (link)). White blood cell counts were read from the peroxidase channel. If necessary, samples were diluted (1:3, 1:8). Platelet counts are not displayed in the results section, since lithium-heparin anticoagulated blood is more prone to platelet clumping and might bias outcome (Nelles and Chandler, 2014 (link)).
Additional blood was collected in 9 ml serum and ethylenediaminetetraacetic acid tubes (Sarstedt, Nümbrecht, Germany). Tubes were centrifuged according to the manufacturer’s instructions and immediately frozen (−80°C) until analysis. Parameters of endothelial matrix remodeling (MMP-2 and -9; TIMP-1) were determined by ELISAs (R&D Systems Europe, United Kingdom). MMP activity was analyzed using Fluorogenic Peptide Substrate (R&D Systems Europe, United Kingdom) (Bernecker et al., 2011 (link)). Parameters of hematopoietic stem cell mobilization (SDF-1α; IL-3) were measured by Procarta multiplex cytokine kit (Affymetrix, Santa Clara, CA, United States) (Schafer et al., 2016 (link)). Inter- and intra-assay CVs for ELISAs were below 15%.

Blood was drawn between 8 and 10 a.m. on the day of testing. Study participants were instructed to fast for at least 8 h prior to the blood draw and this was verified by a questionnaire on the day of testing. To obtain plasma for the Olink panel, blood was drawn into ethylenediaminetetraacetic acid tubes (Sarstedt, Switzerland), centrifuged for 15 min at 1500 × g and plasma was frozen at −80 °C. hsCRP was measured in serum: blood was collected into a silica and gel containing tube (BD Vacutainer).