Heparin

Frozen vials with primary AML cells were rapidly thawed in a 37 °C waterbath. The thawed cell suspension was added dropwise to 10 mL of the RPMI-1640 medium (Gibco^TM Thermo Fisher Scientific, Waltham, MA, USA) containing heparin (20 U/mL; Biochrom AG, Berlin, Germany), DNAse I (8 U/mL; Sigma-Aldrich^®, St. Louis, MO, USA) and MgCl₂ (4 mM; home-made) and incubated at 37 °C for 1 h. Next, cells were pelleted for 10 min at 300× g and resuspended in the culture medium. The cells were incubated overnight to allow cytokine-mediated activation and expansion before proceeding to B/T cell depletion. The cell viability was measured via trypan blue exclusion assay right after thawing as well as after the overnight cytokine induction.

N. meningitidis strain MC58 or its isogenic mutant lacking NHBA (MC58ΔNHBA) was harvested from overnight growth on Columbia sheep blood agar plates and resuspended in KBR buffer (Virion\Serion) with 0.1% BSA and adjusted to OD 0.1. In a conical 96well plate (Sarstedt), 10 µl of FITC-heparin (ThermoFisher H7482) was added to 100 µl bacterial suspension, resulting in 100 µg/ml FITC-heparin. Where indicated, a 20-fold excess of heparin (2000 µ/ml final concentration; Biochrom) was added. Samples were incubated 30 min at room temperature. For ‘bound FITC-heparin’, samples were washed as above with cold KBR + 0.1% BSA, whereas washing was omitted for ‘free FITC-heparin’ samples. Bacteria were pelletted and resuspended in 4% formaldehyde solution in PBS for 30 min before analysis on a FACScalibur flow cytometer.

Non-immortalized adult HBMEC (ACBRI 376, Cell Systems, Kirkland, WA, USA) were grown to confluence in gelatin (Serva Electrophoresis, Heidelberg, Germany) coated T-75 culture flasks (Greiner Bio-One, Frickenhausen, Germany) in a humid atmosphere at 37 °C with 5% CO₂. Cells were propagated in RPMI-1640 medium (Sigma-Aldrich) supplemented with 10% fetal calf serum (Thermo Fisher Scientific, Waltham, MA, USA), 10% Nu-Serum (BD Biosciences, San Jose, CA, USA), 2 mM L-glutamine, 1 mM sodium pyruvate, 1% minimum essential medium with non-essential amino acids (all: Thermo Fisher), 5 U/mL heparin (Biochrom, Berlin, Germany), and 0.3% endothelial cell growth supplement (Cell Systems). As described previously [26 (link)], confluent monolayers were expanded, and recently thawed cells of passage 8 were used for all experiments. Previous experiments had confirmed basic endothelial characteristics [26 (link)].

Pancreatic islets were isolated by collagenase digestion (0.7 mg/ml) (Sigma-Aldrich Chemie GmbH) and discontinuous Ficoll density gradient. Single-cell suspensions of pancreatic islets and lymphoid tissues were prepared using 70 μm cell strainers (Becton Dickinson, San Diego, CA, USA) and Hank’s buffer [1 x HBSS, 5% (v/v) FCS, 10mM HEPES; all Invitrogen]. Single cell suspensions from spleen were additionally subjected to red blood cell lysis (erythrocyte lysis buffer EL, Qiagen). Peripheral blood mononuclear cells (PBMCs) were obtained by retro-orbital sinus puncture [PBS supplemented with 10% (v/v) Heparin (Biochrom AG, Berlin, Germany)] and Ficoll (VWR, Darmstadt, Germany) gradient centrifugation. mAbs to CD3 (145-2C11), CD4 (RM4-5, GK1.5), CD8 (53–6.7), CD25 (PC61, 7D4), CD44 (IM7), CD62L (MEL-14), Vβ-4 (KT4), and CD49b (R1-2) were purchased from eBioscience (Frankfurt, Germany) or BD (Heidelberg). The samples were analyzed using a LSRII or sorted on a FACS Aria (all BD). Data were analyzed with the FlowJo software (Tree Star).

Immortalized human brain microvascular endothelial cells (HBMEC) were kindly provided by K. S. Kim (Stins et al., 1997 (link)) and were cultured as described previously (Unkmeir et al., 2002 (link)). Briefly, cells were cultured in RPMI-1640 medium supplemented with 1% sodium pyruvate (1 mM), 1% L-glutamine (2 mM), 1% non-essential amino acids (all purchased from GE Healthcare, Little Chalfont, United Kingdom), 5 U/ml heparin (Biochrom, Berlin, Germany) and 30 μg/ml endothelial cell growth supplement (ECGS, CellSystems, Troisdorf, Germany). Cells were incubated at 37°C and 5% CO₂ in a humidified atmosphere.

Primary human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cords donated by patients giving birth in the “Marienkrankenhaus Luebeck” (approved by Local Ethics Committee Case: 18-325), as described before [18 (link)]. Cells were cultured in standard cell culture media culture medium (Gibco Medium 199 + Fetal Calf Serum 10% (Gibco, Carlsbad, CA, USA) + Penicillin/Streptomycin 1% (Gibco, Carlsbad, CA, USA) + Large Vessel Endothelia Supplement 1% (Gibco, Carlsbad, CA, USA) + Heparin 5000 U/mL (Biochrom, Schaffhausen, Switzerland). HUVECs were used until passage 3 for experiments. Primary human aortic endothelial cells (HAEC) were obtained from Innoprot (Derio, Spain) and cultured according to the manufacturer’s instructions. When indicated, cells were exposed to control or uremic human sera for five days while treated or not with MR inhibitor spironolactone (1 µM) or ENaC inhibitor amiloride (10 µM). HAECs were used until passage 5 for experiments.

Nalm-6 cells and Molm-13 were purchased from DSMZ (Braunschweig, Germany), and cultured in RMPI medium containing 10% FCS in addition to 100 μg/mL penicillin/streptomycin, 1 mM sodium pyruvate, 1% non-essential amino acids and 2 mM N-acetyl-L-alanyl-L-glutamine (Sigma Aldrich, Darmstadt, Germany). The cells were used without further authentication. Nalm-6 cells and Molm-13 cells were transduced to express the firefly luciferase (Photinus pyralis) using a lentiviral vector, resulting in Nalm-6 Luc and Molm-13 Luc, as described previously [50 (link)]. TM-producing 3T3 cell lines were cultured in DMEM medium, containing 10% FCS, 100 μg/mL penicillin/streptomycin and 1% non-essential amino acids (Sigma Aldrich). Cells were kept at 37 °C with 5% CO₂ and passaged twice per week.
Patient-derived frozen bone marrow mononuclear cells (BMNCs) were quickly thawed and then incubated for 1 h at 37 °C in RPMI 1640 medium, containing 5% FCS, 2500 U/mL Heparin (Biochrom GmbH) and 2000 U/mL DNAse (Sigma Aldrich). Afterwards, the cells were cultured in StemSpan^TM medium (STEMCELL Technologies GmbH, Cologne, Germany), containing 2% FCS, 100 μg/mL penicillin/streptomycin, 2 mM N-acetyl-L-alanyl-L-glutamine, and supplemented with 10 ng/mL of fms-related tyrosine kinase 3 ligand (FLT3-L), 10 ng/mL stem cell factor (SCF), 10 ng/mL thrombopoietin (TPO) and 10 ng/mL interleukin-3 (IL-3).