Blood was collected from healthy volunteers after fasting state of at least 3 hours. The venous blood samples were taken from healthy volunteers, of 24 (mean 3 SD) years of age in tubes containing sodium citrate in the ratio of 9:1 (vol per vol). Sodium nitrite (NaNO2), adenosine 5′diphosphate (ADP), collagen and Dextran 500 were purchased from Sigma (St Louis, MO, USA). DETA‐NONOate (diethylenetriamine NONOate) was purchased from Cayman Chemical (Ann Arbor, MI, USA). RRx‐001 was obtained from EpicentRx (San Diego, CA, USA). The nitric oxide inhibitor 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazole‐1‐oxyl 3‐oxide (Carboxy‐PTIO) was purchased from Sigma (St Louis). Luciferin‐luciferase reagent (Chrono‐Lume) was purchased from Chrono‐Log Corporation (Howertown, PA, USA). DETA‐NONOate was freshly prepared by dissolving in 0.01 mol/L NaOH and used within 1 day. Immediately before use, DETA‐NONOate was diluted in phosphate‐buffered saline (PBS, pH 7.4). Sodium nitrite, ADP and Carboxy‐PTIO were prepared in PBS at pH 7.4. Collagen was prepared in deionized water. Monoclonal antibodies, FITC‐labelled anti‐human CD41a and PE‐labelled anti‐human CD62P, were purchased from Becton Dickinson (San Jose, CA, USA).
Dextran 500
Manufactured by Merck Group
Sourced in United States
Dextran 500 is a high-molecular-weight polysaccharide derived from the fermentation of sucrose by the bacterium Leuconostoc mesenteroides. It is a common laboratory reagent used as a volume expander and for various chromatographic applications.
Automatically generated - may contain errors
Lab products found in correlation
Peg 8, by Merck Group (2 mentions)
Ficoll 70, by GE Healthcare (2 mentions)
Polyethylene glycol 3350, by Merck Group (1 mentions)
Fibronectin from human plasma, by Merck Group (1 mentions)
Endothelial growth medium 2 egm 2, by Lonza (1 mentions)
Ficoll paque, by Cytiva (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Biotwin cm120 transmissionelectron microscope, by Philips (1 mentions)
Hank s balanced salt solution, by Corning (1 mentions)
Ficoll paque, by Thermo Fisher Scientific (1 mentions)
Chondroitinase abc, by Merck Group (1 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (1 mentions)
Bodipy fl gdp, by Thermo Fisher Scientific (1 mentions)
P nitrophenyl phosphate pnpp, by Merck Group (1 mentions)
Bovine serum albumin bsa, by GE Healthcare (1 mentions)
Prostaglandin e1 pge1, by Merck Group (1 mentions)
Horm collagen, by Takeda Pharmaceuticals (1 mentions)
U46619, by Merck Group (1 mentions)
Percoll, by Merck Group (1 mentions)
Citric acid, by Merck Group (1 mentions)
20 protocols using dextran 500
1
Platelet Function Assay with Nitric Oxide
2
Isolation and Purification of Membrane Fractions
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MF were obtained from homogenized leaves as described [22 (link)]. PM was purified from MF and separated by partitioning in two-phase aqueous polymer systems using dextran 500 (Sigma-Aldrich, St. Louis, MO, USA) and polyethylene glycol 3350 (Sigma-Aldrich, St. Louis, MO, USA) as described in [22 (link)]
VM or tonoplast were purified by fractionation of MF by free flow zonal electrophoresis (FFZE) using the BD FFE system (BD Proteomics, Munich, Germany), as described [23 (link)].
VM or tonoplast were purified by fractionation of MF by free flow zonal electrophoresis (FFZE) using the BD FFE system (BD Proteomics, Munich, Germany), as described [23 (link)].
3
Engineered 3D Microvascular Models
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The design and fabrication of three-dimensional in vitro “endothelialized” microvasculature models were described previously (Tsai et al., 2012 (link)). The microchannels were coated with 50 μg/ml fibronectin from human plasma (Sigma) and then incubated at 37°C, 5% CO2, for 1 hr. Endothelial cells were prepared at a concentration of 500,000 cells/ml in Endothelial Growth Medium-2 (EGM-2; Lonza) with 8% mass/vol of dextran 500 (Sigma). Endothelial cells were manually preloaded into the inlet tubing and infused into the channels with a syringe pump at 1.25 ml/min for 2 hr, and then perfused with EGM-2 at the same rate for 2–3 days. Live or cryo MSCs were labeled with carboxyfluorescein succinimidyl ester to render them fluorescent and then suspended at 1 × 106 cells/ml in EGM-2. Following syringe loading, the MSCs were pumped through the microchannels for 1 hr at 1.25 ml/min. Epifluorescence and bright-field microscopy were acquired following the experiment using an EVOS FL Cell Imaging System.
4
Isolation of human polymorphonuclear leukocytes
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The trial was approved by the Ethics Committee of the Medical University of Vienna (#455/2010). Human polymorphonuclear leukocytes (PML) were isolated from peripheral blood of healthy donors, as previously described.32 (link) Briefly, fresh venous blood was loaded on Ficoll-Paque (Amersham Pharmacia Biotech, Uppsala, Sweden) and centrifuged at 350 × g for 35 min. PMLs were collected and separated from the red blood cells by 6% dextran 500 (Sigma Aldrich) sedimentation, followed by hypotonic lysis of the remaining erythrocytes.
5
Ultrastructural Characterization of Samples
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Samples before and after TDP treatment were stored in 20% dextran-500 (Sigma Aldrich, St. Louis, MO, USA) phosphate buffered saline (PBS) (Gibco) overnight to minimize any swelling effects before fixation. The fixative contained 3% paraformaldehyde, 1.5% glutaraldehyde, 5 mM MgCl2, 5 mM CaCl2, 2.5% sucrose, and 0.1% tannic acid in 0.1 M sodium cacodylate buffer, at pH 7.2. Samples were fixed overnight at 4 °C, then post-fixed in 1% osmium tetroxide, followed by staining with 2% aqueous uranyl acetate and dehydrated in increasing concentrations of ethanol (from 30% to 100%). Thin sections (60–90 nm) were cut and stained with lead and uranyl acetate. Sections were observed with Philips/FEI BioTwin CM120 Transmission Electron Microscope at 80 kV.
6
Profiling immune cell responses to S. aureus
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Neutrophils and peripheral blood mononuclear cells (PBMCs) were isolated from collected fasting blood samples. Briefly, after Ficoll-Paque (Fisher) centrifugation of peripheral blood, neutrophils and PBMCs were separated from erythrocytes by 3% dextran-500 (Sigma) density-gradient sedimentation. Isolated cells were resuspended in Hank’s Balanced Salt Solution (Cellgro) for cell counts. The in vitro assays described below were used to measure functional responses to S. aureus. Isolated PBMC and neutrophil cell counts were expressed as cell count × 107.
7
Ex Vivo Keratoconus Model for TDP Analysis
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Fresh albino rabbit eyes were purchased from Pelfreez Biologicals (Rogers, AR, USA). The ectatic model was previous developed in our lab [21 (link)], briefly, to create the ex vivo keratoconus-like model (KC) corneas, the eyes were treated with 0.1 U/mL chondroitinase ABC (C36675U Sigma Aldrich, St. Louis, MO, USA) by immersing the anterior surface in enzyme solution for 2 h. Following enzyme treatment, the eye globes were washed with full medium several times. The KC corneas were then treated to the different TDPs by submersing the anterior segment in TDP-suspended medium containing 4% dextran-500 (Sigma Aldrich, St. Louis, MO, USA) for 18 h. The dextran was added to reduce extensive swelling of the ex vivo corneas. Control corneas without enzyme treatment were cultured in 4% dextran full medium without TDP microparticles.
8
Platelet Activation Assay Protocol
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Prostaglandin E1 (PGE1), indomethacin, U46619, citric acid, trisodium citrate, ionomycin, glycerol, dextran-500, Nonidet P-40 (NP-40), fibrinogen, p-nitrophenyl phosphate (pNPP) and Percoll® were from Sigma (Poole, U.K.). Ethylene glycol-bis(2-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA) was from Calbiochem (Nottingham, U.K.). BODIPY-FL-GDP, dithiothreitol, fluorescein (FITC)-conjugated anti-CD15 and allophycocyanin (APC)-conjugated anti-CD11b/CD18 antibodies were from Thermo Fisher Scientific, (Loughborough, U.K.). Alexa488-conjugated anti-mouse F(ab)2 was from Jackson ImmunoResearch (Ely, U.K.). Murine IgG2aκ isotype control antibody was from BioLegend (London, U.K.). (RS)-citalopram and PAF were from Cambridge Bioscience (Cambridge, U.K.). Horm® collagen was from Takeda (Linz, Austria). Bovine serum albumin (BSA) was from GE Healthcare (Buckinghamshire, U.K.). Fura-2 (AM) was from TEFLabs (Cambridge, U.K.). CRPXL was synthesised in the laboratory of Professor Richard Farndale (University of Cambridge, U.K.).
9
Crowded Lipid Membrane Reconstitution
10
Preparation of Fluorescent Lipid Vesicles
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Ficoll 70 was from GE healthcare and Dextran 500, PEG 8, GTP nucleotide and other analytical grade chemicals were from Sigma. FITC labelled Dextran 500 (Dextran 500-FITC) was from Molecular Probes/Invitrogen. Polar extract phospholipids from E. coli and lissamine-rhodamine B labelled PE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)), from Avanti Polar Lipids (Alabama, USA), were stored in chloroform at −20 °C.
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