S monovette k3

Blood was collected in 1.2 mL (4 mL for clinical trial A and B) Ethylenediaminetetraacetic acid (EDTA)-containing S-Monovette K3 (Sarstedt # 02.1066.001). Plasma was separated from erythrocytes by single centrifugation as previously applied [21 (link),41 (link)], no further centrifugation was applied to the erythrocytes fraction obtained [42 (link)]. Sample preparation for fatty acid methyl esters (FAME) analysis was done as previously described [21 (link)]. Blood fractions were kept on ice during sample preparation and finally stored at −80 °C until FA analysis. As previously described [21 (link),41 (link)] FAMEs of erythrocytes and plasma were prepared directly in the tubes in which they were aliquoted and stored. Internal standards FAME 21:0 (1 mg/mL) and phosphatidylcholine 23:0 (0.4 mg/mL) or TAG 13:0 (0.1mg/mL) were added (100 µL each) plus 2 mL of methanol, 2 mL of catalyst methanol/HCl (3N) and 1 mL of n-hexane.
Analysis of total FAMEs was performed by Fast Gas Chromatography (GC), as previously described [21 (link)] on a 7890 Agilent gas chromatograph (Agilent Technologies, Palo-Alto, CA, USA), equipped with a fused-silica BPX-70 capillary column (10 m, 0.1 mm i.d., 0.2-µm film thickness; Spencer Group Engineering, Melbourne, Australia).

Cord blood was collected into S-Monovette^® K3 ethylene diamine tetraacetic acid (EDTA, Sarstedt, Nümbrecht, Germany). Cord blood mononuclear cells (CBMCs) were prepared from the whole blood through density gradient centrifugation using the Lymphocyte Separation Medium 1.077, FicoLite-H (Linaris biological products, Dossenheim, Germany). Whole blood was diluted 1:1 with D-PBS followed by layering the mixture 1.5:1 on FicoLite-H. Samples were centrifuged at 400× g for 30 min. Interphase was considered as CBMCs and were separated. Cells were washed twice using Dulbecco’s phosphate-buffered saline (D-PBS, Sigma Aldrich, Steinheim, Germany) and were centrifuged at 400× g for 10 min. Subsequently, CBMCs were resuspended in fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, MA, USA) + 10% dimethylsufoxid (DSMO, Sigma Aldrich, Steinheim, Germany) and were placed in a CellCamper^® for cryopreservation at −80 °C.