SPR was performed on a GE Healthcare Biacore T200 with a running buffer containing 20 mM HEPES, pH 7.5, 100 mM NaCl and 0.5% Tween-20, with a flow rate of 30 μL/min at 25 °C. A carboxymethylated dextran (CM5) chip (GE Healthcare) was activated with N-hydroxysulfosuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). We then either quenched the CM5 surface with ethanolamine (yielding a blank flow cell) or immobilized HCoV-NL63 S before quenching. 10 μg of HCoV-NL63 S was diluted into 10 mM sodium acetate, pH 5.5 and was directly immobilized for 700 s, thus yielding 28,000 RUs. After immobilization quenching, running buffer was flowed for 10 min to ensure a steady baseline before experimental binding. Heparan sulfate (Sigma Aldrich) was reconstituted in running buffer at 5.0 mg/mL. Two concentrations of Heparan sulfate, 5.0 mg/mL and 2.5 mg/mL, were injected for 80 s with a dissociation time of 400 s. All data were subtracted from the blank flow cell, to account for any nonspecific interactions of Heparan sulfate with the CM5 chip, and the baseline was normalized to 0.
Heparan sulfate
Manufactured by Merck Group
Sourced in United States
Heparan sulfate is a type of glycosaminoglycan found in the extracellular matrix and on the surface of many cells. It plays a crucial role in various biological processes, such as cell signaling, cell-cell adhesion, and growth factor regulation.
Automatically generated - may contain errors
Lab products found in correlation
Heparin, by Merck Group (5 mentions)
Chondroitin sulfate, by Merck Group (4 mentions)
Epidermal growth factor (egf), by Merck Group (4 mentions)
B 27 supplement without vitamin a, by Thermo Fisher Scientific (3 mentions)
Protamine sulfate, by Merck Group (3 mentions)
Basic fibroblast growth factor (bfgf), by Merck Group (3 mentions)
Human fibrinogen, by Enzyme Research (3 mentions)
Stemline medium, by Merck Group (2 mentions)
A6964, by Merck Group (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Igf 1, by Merck Group (2 mentions)
Chondroitinase abc, by Merck Group (2 mentions)
Dermatan sulfate, by Merck Group (2 mentions)
Recombinant human epidermal growth factor and basic fibroblast growth factor, by Thermo Fisher Scientific (2 mentions)
Del a enhancement solution, by PerkinElmer (2 mentions)
Streptavidin europium, by PerkinElmer (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Ultra low cluster plate, by Corning (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Matrigel, by Corning (2 mentions)
31 protocols using heparan sulfate
1
SPR Analysis of HCoV-NL63 S Binding
2
Differentiation of Lgr5+ Progenitors and Spheres
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The flow-sorted cells were diluted to 2 cells/μl in DMEM/F12 medium with 1% N2 (Invitrogen, 17502-048), 2% B27 (Invitrogen, 17504-044), EGF (20 ng/ml; Sigma, E9644), IGF (50 ng/ml, Sigma, I8779), heparan sulfate (20 ng/ml, Sigma, H4777), β-FGF (10 ng/ml, Sigma, F0291), and 0.1% ampicillin (Sigma, A9518-5G) and cultured in Costar ultra-low attachment dishes (Costar, 3599) for 5 days and then passaged to the next generation.
For the differentiation assay, we differentiated both flow-sorted cells and spheres. In the cell-differentiation assay, the flow-sorted Lgr5+ progenitors and Lgr5- SCs were cultured to a density of 50 cells/μl on laminin-coated plates using DMEM/F12 medium with 1% N2 (Invitrogen, 17502-048), 2% B27 (Invitrogen, 17504-044), EGF (20 ng/ml; Sigma, E9644), IGF (50 ng/ml, Sigma, I8779), heparan sulfate (20 ng/ml, Sigma, H4777), β-FGF (10 ng/ml, Sigma, F0291), and 0.1% ampicillin (Sigma, A9518) for 10 days. EdU (10 μM, Invitrogen, C10340) was added during the culture in order to label the dividing cells. In the sphere-differentiation assay, the spheres were plated on laminin-coated four-well dishes and cultured in DMEM/F12 medium with 1% N2 (Invitrogen, 17502-048), 2% B27 (Invitrogen, 17504-044), and 0.1% ampicillin (Sigma, A9518) for 10 days.
For the differentiation assay, we differentiated both flow-sorted cells and spheres. In the cell-differentiation assay, the flow-sorted Lgr5+ progenitors and Lgr5- SCs were cultured to a density of 50 cells/μl on laminin-coated plates using DMEM/F12 medium with 1% N2 (Invitrogen, 17502-048), 2% B27 (Invitrogen, 17504-044), EGF (20 ng/ml; Sigma, E9644), IGF (50 ng/ml, Sigma, I8779), heparan sulfate (20 ng/ml, Sigma, H4777), β-FGF (10 ng/ml, Sigma, F0291), and 0.1% ampicillin (Sigma, A9518) for 10 days. EdU (10 μM, Invitrogen, C10340) was added during the culture in order to label the dividing cells. In the sphere-differentiation assay, the spheres were plated on laminin-coated four-well dishes and cultured in DMEM/F12 medium with 1% N2 (Invitrogen, 17502-048), 2% B27 (Invitrogen, 17504-044), and 0.1% ampicillin (Sigma, A9518) for 10 days.
3
Myogenic Differentiation of iPSCs
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Differentiation of myogenic cells from human iPSCs was performed basically as previously published using a spheres-based culture21 (link). Briefly, hiPSCs were cultured in suspension using ultra low attachment flasks in Stemline medium (Sigma, SA3194) containing 100 ng/ml FGF-2, 100 ng/ml EGF and 5 ng/ml heparan sulfate (Sigma, H7640-1mg). Cells were split weekly using a 2 min accutase (Sigma, A6964) digestion and media were exchanged every second day. After 6 weeks of cultivation, cells were transferred into single cells using accutase digestion and filtration through a 30 μm pre-separation filter (Miltenyi Biotec, 130-041-407). 200,000 Cells per well were seeded on glass coverslips in 24 well plates, coated with PLO and laminin. Medium was changed to DMEM (Gibco, 41965) containing 2% B27 and 1% Antibiotic-Antimycotic with a media exchange twice per week. After 8 weeks of final differentiation mature myotubes were obtained and fixed or lysed for analysis.
To perform co-cultivation of iPSC-derived muscle cells with iPSC-derived motor neurons, myogenic cells were differentiated using Pax7-induced stem cell-derived progenitors, as described22 (link),43 (link).
To perform co-cultivation of iPSC-derived muscle cells with iPSC-derived motor neurons, myogenic cells were differentiated using Pax7-induced stem cell-derived progenitors, as described22 (link),43 (link).
4
Isolation and Cultivation of Murine Cochlear Progenitors
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For each experiment, cochleae of 4–6 neonatal C57BL/6 or Atoh1-nGFP pups47 (link) that express GFP under the control of the Atoh1 enhancer (a generous gift from Jane E. Johnson, University of Texas) were dissected in HBSS and the organ of Corti was separated from the stria vascularis and the spiral ganglion neurons. The tissues were dissociated in trypsin (0.05%) for 13 min in PBS at 37 °C. 10% FBS in DMEM-high glucose medium was used to stop the reaction. After washing, the tissue was manually dissociated. The triturated cells were then passed through a 70 μm cell strainer (BD Labware) to remove tissue debris. Single cells were cultured in DMEM/F12 (1:1) supplemented with N2 and B27 (Invitrogen), and EGF (20 ng/ml; Chemicon), bFGF (10 ng/ml; Chemicon), IGF-1 (50 ng/ml;Chemicon), and heparan sulfate (50 ng/ml; Sigma). Single cells were maintained in ultra-low cluster plates (Costar) for several days in culture to obtain neurospheres. For passage, neurospheres of the first generation were dissociated with a 27G needle and syringe (BD Labware) 6–8 times. Single cell suspensions were cultured in fresh medium F12/DMEM (1:1) with the same growth factors to form neurospheres until use at the 4th to 5th generation.
5
Glycosaminoglycans Characterization Protocol
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The following materials were purchased from the manufacturers indicated: heparin, heparan sulfate, chondroitin sulfate A, B and C, heparinases I and III, chondroitinase ABC, fluorescein isothiocyanate (FITC), GenElute PCR clean-up kit, phospholipase C phosphatidylinositol-specific (PI-PLC) from Bacillus cereus, all from Sigma-Aldrich (St. Louis, MO, USA); 2-O, 6-O and N-desulfated heparins from Amsbio (Abingdon, UK); Dulbecco’s Modified Eagle’s minimal essential medium (DMEM) and Minimum Essential Medium (MEM), fetal bovine serum, penicillin-streptomycin, and PBS-phosphate-buffered saline from Gibco-Thermo Fischer Scientific (Waltham, MA, USA); Brain-Heart Infusion broth from Pronadisa (Madrid, Spain); RNeasy Kit and RNase-Free DNase from Qiagen (Hilden, Germany); High-Capacity cDNA Reverse Transcription Kit and PowerSYBR Green PCR Master Mix from Applied Biosystems (Foster City, CA, USA). Synthetic peptides were from Abyntek Biopharma (Derio, Spain); mouse monoclonal anti-syndecan 1 (CD138) from DakoCytomation (Carpinteria, CA, USA); and rabbit anti-syndecan 2, goat anti-syndecan 3 and rabbit anti-syndecan 4 polyclonal antibodies from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other chemicals were obtained from commercial sources and were of analytical grade.
6
3D Neurosphere Culture with GSK3β Inhibitor
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Two thousand FACS-sorted cells were plated per well into round bottom U-shaped low-adherent 96-well plates (Costar) in DMEM-F12 supplemented with B27, N2 (all from Life Technologies), 20 ng/ml EGF, 10 ng/ml bFGF, 50 ng/ml IGF, and 50 ng/ml heparan sulfate (Sigma-Aldrich). Two–three days after sort, Matrigel (GF-depleted, Corning) was added at a final concentration of 2%. Cells were incubated for 1 week at 37 °C and subsequently transferred, to low-adhesion 24-well plates (Costar) in medium with 2% Matrigel. The GSK3β inhibitor CHIR99021 (3 μM) was added to the culture at two time points (d5/6 and d10/11) together with supplementation of fresh medium.
7
Heparan Sulfate-Mediated IFNγ Binding Assay
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Five microgram of heparan sulfate (Sigma-Aldrich, St Louis, MO) was added into each well of 96-well plates at a volume of 100 µl/well for 2 hours at 37 °C. Wells were added with serially diluted supernatants of COS-7 cells transfected with pCMV-IFNγ or pCMV-IFNγ-HBDn. After incubation for 2 hours at room temperature, each well was washed with phosphate-buffered saline (PBS) −0.05% Tween 20, and proteins bound to heparan sulfate were detected by enzyme-linked immunosorbent assay (ELISA) using antimouse IFNγ antibody (Ready-SET-GO! Murine IFNγ ELISA; eBioscience, San Diego, CA).
8
Fluorescence-based Cell Adhesion Assay
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All chemicals and reagents were
of analytical grade and used without further purification. Common
chemicals were obtained from Fischer Scientific (Pittsburgh, PA) or
Sigma-Aldrich (St. Louis, MO) unless specified. Rink amide 4-methylbenzylhydrylamine
(MBHA) resin, amino acids [Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, and Fmoc-Lys(N3)-OH], Boc-aminooxyacetic acid, Fmoc-ε-Ahx-OH, and HBTU
were purchased from Anaspec (San Jose, CA). Antibodies antipaxillin
and antivinculin and Cy-2 goat anti-mouse IgG were purchased from
BD Biosciences (San Jose, CA) and Jackson ImmunoResearch Laboratories,
Inc. (West Grove, PA), respectively. Fluorescent dyes DAPI and phalloidin
and penicillin/streptomycin were obtained from Invitrogen (Carlsbad,
CA). Fluorescence mounting medium was purchased from Dako (Carpinteria,
CA). Swiss albino 3T3 mouse fibroblasts were obtained from the UNC-CH
Tissue Culture Facility (Chapel Hill, NC). Heparan sulfate, heparinase
I and II, and chondroitinase ABC were obtained from Sigma-Aldrich
(St. Louis, MO).
of analytical grade and used without further purification. Common
chemicals were obtained from Fischer Scientific (Pittsburgh, PA) or
Sigma-Aldrich (St. Louis, MO) unless specified. Rink amide 4-methylbenzylhydrylamine
(MBHA) resin, amino acids [Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, and Fmoc-Lys(N3)-OH], Boc-aminooxyacetic acid, Fmoc-ε-Ahx-OH, and HBTU
were purchased from Anaspec (San Jose, CA). Antibodies antipaxillin
and antivinculin and Cy-2 goat anti-mouse IgG were purchased from
BD Biosciences (San Jose, CA) and Jackson ImmunoResearch Laboratories,
Inc. (West Grove, PA), respectively. Fluorescent dyes DAPI and phalloidin
and penicillin/streptomycin were obtained from Invitrogen (Carlsbad,
CA). Fluorescence mounting medium was purchased from Dako (Carpinteria,
CA). Swiss albino 3T3 mouse fibroblasts were obtained from the UNC-CH
Tissue Culture Facility (Chapel Hill, NC). Heparan sulfate, heparinase
I and II, and chondroitinase ABC were obtained from Sigma-Aldrich
(St. Louis, MO).
9
Bacterial Culture and Protein Purification
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Luria–Bertani (LB)
broth, Luria agar, and kanamycin were purchased from Himedia, India.
Sodium chloride, imidazole, boric acid, Tris–HCl buffer, EDTA,
ethanol, and the Amicon Ultra 10K device were purchased from Merck
(Darmstadt, Germany). Heparan sulfate, sodium dodecyl sulfate, Triton
X-100, and 3,30-diaminobenzidine (DAB) were bought from Sigma, Saint
Louis. All other chemicals used during the experiments were of molecular
biology grade.
broth, Luria agar, and kanamycin were purchased from Himedia, India.
Sodium chloride, imidazole, boric acid, Tris–HCl buffer, EDTA,
ethanol, and the Amicon Ultra 10K device were purchased from Merck
(Darmstadt, Germany). Heparan sulfate, sodium dodecyl sulfate, Triton
X-100, and 3,30-diaminobenzidine (DAB) were bought from Sigma, Saint
Louis. All other chemicals used during the experiments were of molecular
biology grade.
10
Quantifying Anticoagulant Activity of Heparan Sulfate
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The anticoagulant activity of heparan sulfate or heparin derivatives was measured using the Anti-Xa heparin test kit (Iduron, Cheshire, UK) according to the manufacturer’s protocol. heparan sulfate (Sigma-Aldrich) or desulfated heparin derivatives (Iduron) were incubated with anti-thrombin solution (0.5 IU/mL) for 2 min at 37 °C. Cells were treated with Factor Xa (2.5 μg/mL) for 2 min at 37 °C. Factor Xa substrate (0.625 mg/mL) was added to develop color and the reaction was stopped with 20% acetic acid. The absorbance was measured at 405 nm using a microplate reader (Bio-Rad).
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