Heparin sodium salt

Immortalised human brain microvascular endothelial cells (I-HBMEC) (Innoprot, Bizkaia, Spain) (P10361-1M), Immortalised human astrocytes (IA) (Innoprot, Bizkaia, Spain) (P10251-1M), CLTH/Immortalised Pericytes (IP) (Amsbio, Oxfordshire, U.K.) (CL05008-CLTH), D-MEM/F:12 (1:1) (CE) (Thermofisher, Cambridge, U.K.) (11320074), Insulin-trans-sel-G, 100× (Thermofisher, Cambridge, U.K.) (41400045), Penicillin streptomycin (PS) (Gibco, Paisley, U.K.) (15070-063), Fetal bovine serum (FBS) (Gibco, Paisley, U.K.) (A3160801), Hydrocortisone solution (50 um) (Merck, Dorset, U.K.) (H6909), Fibroblast growth factor-basic (BFGF) (Merck, Dorset, U.K.) (SRP3043), Heparin sodium salt (Merck, Dorset, U.K.) (H3149), Dulbecco’s phosphate buffered saline, M (DPBS) (Merck, Dorset, U.K.) (D8537), Accutase^® solution (Merck, Dorset, U.K.) (A6964), Tissue culture flask, 75 cm² growth area (T75) (Sarstedt, Leicestershire, U.K.) (83.3911.002).

For each aNPC preparation, three adult (3-4-month-old) male mice were humanely euthanized. The brains were extracted and hippocampi were isolated under a dissecting microscope using fine surgical instruments and collected in ice-cold PIPES buffer pH 7.4 containing 20 mM PIPES, 25 mM glucose, 0.5 M KCl, 0.12 M NaCl (Sigma, St. Louis, MO), and 100 U/100 μg/mL Penicillin/Streptomycin solution (Invitrogen, Carlsbad, CA). After centrifugation (110 ×g × 5 min), tissue was digested for 40 minutes at 37°C using the Papain Dissociation System (Worthington DBA, Lakewood, NJ). The cell suspension was placed into 25 cm² Falcon cell-culture flasks (Thermo Fisher Scientific, Rockville, MD) and cultured in growth medium [Neurobasal-A medium containing B27 supplement, 2 mM L-glutamine (Invitrogen), 10 ng/mL human basic fibroblast growth factor (bFGF-2, PeproTech, Rocky Hill, NJ), 20 ng/mL human epidermal growth factor (EGF, Sigma), 4 μg/mL heparin sodium salt (Sigma), and 100 U/100 μg/mL Penicillin/Streptomycin]. Primary (Passage 1, P1) neurospheres were passaged after 7–9 days in vitro (DIV), whereas subsequent passages were prepared every 5 DIV. At each passage, cells were plated in a T25 flask at a density of 12,000 cells/cm² in growth medium. P3–P10 neurospheres were utilized for experiments.

Recombinant human basic fibroblast growth factor (FGF-2) 146 aa (carrier free, purity better than 97%, lyophilized) was purchased from R&D Systems (Minneapolis, USA). Just before use, it was reconstituted in PBS containing 0.1% of Human serum albumin. Human serum albumin (≥97%) and heparin sodium salt from porcine intestinal mucosa (17–19 kDa), glutaraldehyde (25% aqueous solution), (3-aminopropyl)triethoxysilane (APTES, 99%), fluorescein isothiocyanate isomer I (FITC, ≥90%) were obtained from Sigma-Aldrich. All the salts and organic solvents were of analytical grade. Deionized water (18.2 MΩ, Milli-Q Ultrapure Water System, Millipore) was used in all experiments.
Albumin was conjugated with FITC according to the published protocol[31 ] with small modifications. Briefly, 20 mg of Alb was dissolved in 0.1 M carbonate-bicarbonate buffer (pH 9) to a final concentration of 2.5 mg/mL. The FITC solution (9.5 mg/mL) was added drop-wise to the albumin solution, while stirring. The reaction was carried out 4 h in the dark at room temperature. The product was dialyzed (Spectrapor, cut-off 3.5 kDa) against water. Finally, the Alb^FITC product was lyophilized and the degree of labelling was determined spectroscopically according to the protocol suggested by the supplier (Sigma Aldrich). The molar ratio of FITC to Alb was 0.7.

D-glucuronic acid (G526), 4-methylumbelliferone (M1381), HA sodium salt from Streptococcus equi mol wt 15,000 to 30,000 (97616), 1,6-Hexanediol (H11807), N-acetyl-D-glucosamine (A3286), heparin sodium salt from porcine intestinal mucosa (H3393), D-mannitol (M4125), (3-aminopropyl)trimethoxysilane (281778), and 70% (v/v) glutaraldehyde (G7776) were from Sigma-Aldrich. 2% (w/v) bis acrylamide (1610142) and 40% (w/v) acrylamide solutions (1610140) were from Bio-Rad Laboratories. TEMED (TB0508) was from Bio Basic. Ammonium persulfate (17-1311-01) was from Life Sciences, and hyaluronidase from Streptomyces hyaluronlyticus (389561) was from Millipore Corp. Collagen I (354236) was from BD Bioscience. Sulfo-SANPAH crosslinker (A35395) was from Thermo Fisher Scientific. Recombinant human protein arginine deiminase 4 (PAD4) was from Cayman Chemical, Inc.

The anticoagulant activity was determined with prothrombin time (PT) and activated partial thromboplastin time (APTT) following the standard method described by [21 ]. 250 μL of human platelet-poor-plasma was incubated in a reaction vial with 100 μL of different concentrations of BVME, BVPE, BVAE, TPME, TPPE, TPME, OSME, OSPE, and OSAE (50, 25, and 12.5 mg/mL, respectively) for 7 minutes at room temperature before being subjected to PT and APTT tests.
Neoplastin Cl Plus reagent and STA-PTT automated reagent, Diagnostica Stago (Asnieres-sur-Seine, France), were reconstituted and subjected to PT and APTT assays, together with 0.025M calcium chloride. The PT and APTT assays were analysed using STA Compact coagulation analyser (Diagnostica Stago, France) according to the manufacturer's protocol. The time taken for clot formation was detected using the analyser and compared with the PT and APTT from control (without extracts) as the negative control and plasma mixed with heparin sodium salt (140 IU/mg) (Sigma-Aldrich, USA) as the positive control. All experiments were carried out in triplicate.

Cholesterol, cholic acid, human thrombin (EC: 3.4.21.5), human fibrinogen, O-Phthaldialdehyde reagent (OPA), pepsin (EC: 3.4.23.1), pancreatin (EC: 232-468-9), bile salts, α-amylase (EC: 3.2.1.1), lysozyme (EC: 3.2.1.17), sodium taurocholate, bile assay kit, heparin sodium salt, mucin, bovine serum albumin (BSA), glucoronic acid, trichloroacetic acid, L-leucine, cholestyramine, linoleic acid, trichloroacetic acid, galactose and glucosamine were purchased from Sigma-Aldrich Chemicals Co. (St. Louis, MO, USA). M17 broth, lactose and dextrose were purchased from DIFCO (Sparks, MD, USA). Thrombin time and pro-thrombin time kits were purchased from Wiener Lab (Rosario, Argentina). A Cholesterol assay kit was purchased from RANDOX Laboratories (Crumlin, UK). The DC Lowry protein assay was purchased from Bio-Rad Laboratories (Hercules, CA, USA).