HUVECs were plated on cell culture–grade plastic dishes coated with 0.1% gelatin and grown to confluence. A cone and plate flow apparatus,39 which maintains cells at 5% CO2 and 37°C, was used to induce a shear stress protocol. The applied shear stress protocol consisted of a 24‐hour preconditioning period at a steady shear stress of 15 dyne/cm2, which was then either increased to 30 dynes/cm2 (nonreversed flow) or increased to 30 dynes/cm2 and reversed in direction (reversed flow) to simulate relative hemodynamics previously quantified in our in vivo FAL model.42 Fresh culture media consisting of M199 with 4% dextran from Leuconostoc spp (Sigma‐Aldrich, Mr ≈500 000), 2% fetal bovine serum, 100 U/mL penicillin‐G + 100 μg/mL streptomyocin, 2 mmol/L L‐glutamine, 5 μg/mL EC growth supplement, and 10 μg/mL heparin was added to cells before exposure to shear stress and was continuously exchanged throughout the duration in the cone and plate apparatus.
Dextran from leuconostoc spp
Manufactured by Merck Group
Sourced in China
Dextran from Leuconostoc spp. is a high-molecular-weight polysaccharide produced by the lactic acid bacteria Leuconostoc species. It is a naturally occurring carbohydrate polymer consisting of glucose monomers linked primarily through alpha-1,6 glycosidic bonds. The core function of Dextran is to serve as a versatile and biocompatible material for various laboratory and research applications.
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Lab products found in correlation
Rpmi 1640 media, by Thermo Fisher Scientific (2 mentions)
Optilab rex refractive index detector, by Wyatt Technology (1 mentions)
Dawn heleos 2 multi angle laser photometer, by Wyatt Technology (1 mentions)
Smz25, by Nikon (1 mentions)
Mcdb 131 medium, by Merck Group (1 mentions)
Polyethylene glycol peg 8000, by Merck Group (1 mentions)
6 dodecanoyl 2 dimethylaminonaphthalene laurdan, by Thermo Fisher Scientific (1 mentions)
Sylgard 184 silicone elastomer kit, by Dow (1 mentions)
1 2 dioleoyl sn glycero 3 phosphocholine, by Avanti Polar Lipids (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Sodium hydroxide (naoh), by Merck Group (1 mentions)
Chloroform, by Merck Group (1 mentions)
Atto647n dope, by ATTO-TEC (1 mentions)
Minimum essential medium (mem), by Merck Group (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Gilteritinib, by MedChemExpress (1 mentions)
Glass bottom microwell dishes, by MatTek (1 mentions)
Bovine serum albumin 4 6 diamidino 2 phenylindole dapi, by Merck Group (1 mentions)
Dylight488 maleimide, by Thermo Fisher Scientific (1 mentions)
9 protocols using dextran from leuconostoc spp
1
Shear Stress Induced Endothelial Response
2
Characterization of Arabinoxylan Molecular Weight
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Molecular weight distribution of purified arabinoxylan fractions were determined by gel permeation chromatography with multi-angle static light scattering (GPC-MALLS) [40 (link)]. GPC-MALLS measurement of polysaccharides was performed on a DAWN HELEOS-II multi-angle laser photometer (Wyatt Technology Corporation, California, USA), Optilab rEX Refractive Index Detector (Wyatt Technology Corporation, California, USA) with a LaChrom Elite pump L-2130 (Hitachi, Ltd., Japan) equipped with TSK-GEL G3000 PWxl and G4000 PWxl column (7.8 mm × 300 mm) for aqueous solution. Dextran from Leuconostoc spp. (Mr–40,000, Sigma-Aldrich., Shanghai, China) was injected as the standard twice.
The eluent was sodium chloride aqueous solution (NaCl, 0.1 mol/L) with a temperature of 25 °C and at a flow rate of 0.5 mL/min. All of the samples were dissolved in the eluent with a concentration of 1.0 mg/mL and filtrated with polytetrafluoroethylene syringe membrane filter (0.45 µm pore size, Fisher Scientific, Waltham, USA). The injection volume was 200 µL, and the elution time was 30 min. Astra software was utilized for data acquisition and analysis.
The eluent was sodium chloride aqueous solution (NaCl, 0.1 mol/L) with a temperature of 25 °C and at a flow rate of 0.5 mL/min. All of the samples were dissolved in the eluent with a concentration of 1.0 mg/mL and filtrated with polytetrafluoroethylene syringe membrane filter (0.45 µm pore size, Fisher Scientific, Waltham, USA). The injection volume was 200 µL, and the elution time was 30 min. Astra software was utilized for data acquisition and analysis.
3
Studying Particle Adhesion in Blood Mimics
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To mimic the viscosity of blood, the particles were suspended in dextran solution of 3.5 mPa·sec viscosity (Dextran from Leuconostoc spp., Mr ~40,000, Sigma-Aldrich). The particles were perfused in a closed-circuit loop via a peristaltic pump (Watson Marlow 530C, Watson Marlow, UK) at 200 mL/min flow rate. The solution then entered a custom-made damper (250 mL bottle with a Thermo Scientific Nalgene 3 port filling/venting cap). The main objective of the pump damper is to adsorb pulsations generated in the operation, thus assuring a stable, constant flow rate. To monitor the adhesion the models were placed under a fluorescent upright microscope (Nikon SMZ25, Nikon Instruments Inc., Melville, NY, USA) and time-lapse imaging was performed.
4
Shear Stress Modulation of Endothelial Cells
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Confluent monolayers of HAEC grown on a 6-well plate were exposed to unidirectional pulsatile shear stress (PSS; 6, 12, and 24 h) using a modified flow device (14 (link)). A neutralized MCDB-131 medium (Sigma Aldrich, MO) containing 7.5% sodium bicarbonate solution, 10% FBS, and 4% dextran from Leuconostoc spp (Sigma Aldrich, MO) was used for PSS exposure. Following exposure to PSS, the center of each monolayer was removed by using a cell scraper to collect only flow-aligned cells from the periphery of the well. To visualize changes in endogenous protein expressions and proximity ligations, we utilized our in-house dynamic flow system (∂τ/∂t = 29.3 dyne·cm−2·s−1, with time-averaged shear stress = 50 dyne·cm−1 at 1 Hz) (33 (link)).
5
Fluorescent Lipid Membrane Characterization
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1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) was purchased from Avanti Polar Lipids (IL, USA). 6-acetyl-2-dimethylaminonaphthalene (ACDAN) was purchased from Santa Cruz Biotechnology (USA), and 6-dodecanoyl-2-dimethylaminonaphthalene (LAURDAN) from Thermofisher Scientific (USA). ATTO 647N-DOPE was obtained from ATTO-TEC GmbH (Siegen, Germany). Polydimethylsiloxane (PDMS) and curing agent were obtained as part of the SYLGARD® 184 silicone elastomer kit from Dow Corning (Michigan, USA). Dextran from Leuconostoc spp (Mw 450–650 kg/mol), and poly(ethylene glycol) (PEG 8000, Mw 8 kg/mol) were purchased from Sigma-Aldrich. Chloroform obtained from Merck (Darmstadt, Germany) was of HPLC grade (99.8%). The lipid stocks were mixed as Chloroform solutions at 4 mM, containing 0.1 mol% ATTO 647N-DOPE or 0.5 mol% LAURDAN, and were stored until use at −20 °C. Fluorescein isothiocyanate isomer (FITC), sucrose, glucose, dimethyl sulfoxide (DMSO), sodium hydroxide (NaOH) and sodium chloride (NaCl) were obtained from Sigma-Aldrich (Missouri, USA). All aqueous solutions were prepared using ultrapure water from a SG water purification system (Ultrapure Integra UV plus, SG Wasseraufbereitung) with a resistivity of 18.2 MΩ cm.
6
Preparation and Analysis of Cells
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MEM (Minimum Essential Medium) and Dextran from Leuconostoc spp. were purchased from Sigma-Aldrich (St. Louis, MO). Qubit dsDNA HS assay kit was from Invitrogen (Carlsbad, CA). Ficoll-Paque PLUS was from GE Healthcare (Chicago, IL).
7
Evaluating Hyaluronic Acid and Gilteritinib
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HA (Mw = 20 kDa) was a product of Lifecore Biomedical (Chaska, MN, USA). Gilteritinib (GLT) was acquired from MedChemExpress (South Brunswick, NJ, USA). EGCG was a product of DSM Nutritional Products Ltd. (Heerlen, The Netherlands). Amicon Ultra-15 centrifugal filter devices, NaCl, and urea were obtained from Merck Millipore Corporation (Darmstadt, Germany). Triton X-100, Tween 20, bovine serum albumin (BSA), 4′,6-diamidino-2-phenylindole (DAPI), and dextran from Leuconostoc spp. (Mw = 15–25 kDa) were obtained from Sigma-Aldrich (St. Loius, MN, USA). RPMI 1640 media, DMEM media, penicillin/streptomycin solution, fetal bovine serum (FBS), phosphate-buffered saline (10 mM, pH 7.4, PBS), DyLight 488 maleimide, CellROX Green oxidative stress detection reagent, and Image-iT LIVE Red apoptosis detection kit were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Caspase-Glo 3/7 assay kit and CellTiter-Glo viability assay reagent were obtained from Promega (Madison, WI, USA). Glass-bottom microwell dishes (dish size = 35 mm, thickness = 0.13–0.16 mm) were obtained from MatTek Corporation (Ashland, OR, USA). Float-A-Lyzer tubing (Mw cutoff = 3.5–5 kDa) was a product of Spectrum Laboratories (Piscataway, NJ, USA).
8
Ifosfamide-loaded PLGA Nanoparticles
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Ifosfamide (≥98 %) was purchased from Sigma Aldrich (St. Louis, MO, USA).Poly(d,l-lactic-co-glycolic acid) (PLGA) (Mw: 10,000; lactic acid : glycolic acid = 50:50) was procured from Wako Pure Chemical (Tokyo, Japan). Dextran from Leuconostocspp was also obtained from Sigma-Aldrich (China). All other chemicals were reagent grade and used without further purifications.
9
Shear Stress Induced Endothelial Response
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HUVECs were plated on cell culture grade plastic dishes coated with 0.1% gelatin and grown to confluence. A cone and plate flow apparatus,82 (link) which maintains cells at 5% CO2 and 37°C, was used to induce a shear stress protocol. The applied shear stress protocol consisted of a 24-hr preconditioning period at a steady 15 dyne/cm2, which was then either increased to 30 dynes/cm2 (non-reversed flow) or increased to 30 dynes/cm2 and reversed in direction (reversed flow) to simulate relative hemodynamics previously quantified in our in vivo FAL model.11 (link) Fresh culture medium, consisting of M199 with 4% dextran from Leuconostoc spp (Sigma-Aldrich, Mr ∼500,000), 2% fetal bovine serum, 100 U/mL penicillin-G + 100 μg/mL streptomycin, 2 mmol/L L-glutamine, 5 μg/mL EC growth supplement, and 10 μg/mL heparin, was added to cells before exposure to shear stress, and it was continuously exchanged throughout the duration in the cone and plate apparatus.
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