Cell surface heparan sulfate chains of HAECs were digested by heparinase III as described method (Kerever et al. 2007 (link)) with some modifications. HAECs were incubated with 5 mU/mL of heparinase III (Sigma‐Aldrich, St. Louis, MO) in 50 mmol/L HEPES buffer (pH 7.0), containing 100 mmol/L NaCl and 1 mmol/L CaCl2 at 37°C for 1 h. After the incubation, the heparinase III solution was removed and the cells were cultured with the growth media. We confirmed successful heparinase III digestion using immunostaining. A time course of the eNOS RNA expression was performed using qPCR as described above.
Heparinase 3
Manufactured by Merck Group
Sourced in United States
Heparinase III is a laboratory reagent that is used for the enzymatic degradation of heparin. It is a protein-based enzyme that specifically cleaves heparin, a common anticoagulant, into smaller fragments. The primary function of Heparinase III is to facilitate the analysis and characterization of heparin and heparin-related molecules in various research and diagnostic applications.
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Lab products found in correlation
Chondroitinase abc, by Merck Group (6 mentions)
Heparin, by Merck Group (4 mentions)
Heparinase 1, by Merck Group (4 mentions)
Heparinase 2, by Merck Group (3 mentions)
Neuraminidase, by Merck Group (2 mentions)
Hyaluronic acid, by Merck Group (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Envision 2104 microplate reader, by PerkinElmer (1 mentions)
Alamarblue cell viability reagent, by Thermo Fisher Scientific (1 mentions)
Cd56 v450, by BD (1 mentions)
Anti rabbit igg alexa fluor 647, by Cell Signaling Technology (1 mentions)
Anti rabbit igg hrp, by Cell Signaling Technology (1 mentions)
Heparin, by STEMCELL (1 mentions)
Formyl methionyl leucyl phenylalanine (fmlp), by Merck Group (1 mentions)
Hyaluronidase, by Merck Group (1 mentions)
Background suppressor dye, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342 stain, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Heparinase, by Merck Group (1 mentions)
Sodium chlorate, by Merck Group (1 mentions)
31 protocols using heparinase 3
1
Heparinase III Digestion of HAEC Surface Heparan Sulfate
2
Disrupting Actin Filaments and Reducing Surface Proteoglycans in HeLa Cells
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HeLa cells were cultured in Dulbecco’s Modified Eagle’s medium with 10% (v/v) fetal bovine serum without phenol red. Before performing the single-particle tracking, we plated the cells in a slide with eight-well chambers. Once a 70%–80% confluence had been reached, the HeLa cells were deprived of serum for 24 h. To disrupt their actin filaments, the cells were pretreated with a culture medium containing 10 M cytochalasin D (Cyto D) for 1 h. Fluorescence-activated cell sorting (FACS) showed that when HeLa cells were treated with 10 mU of HS lyase [16 (link)] for 2 h at 37 C, the surface proteoglycans (PGs) can be reduced almost to background levels. Therefore, to achieve a partial removal of surface PGs, we added Heparinase III (Sigma-Aldrich, St. Louis, MO, USA) to a culture medium at a final concentration of 5 mU/mL and incubated the cell culture for 1 h at 37 C, which reduces the surface PGs to less than 30% of the native value.
3
Dopaminergic Neuron Differentiation Assay
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For in vitro assays, DAPs at day 28 were dissociated, replated at a density 3.0 × 104 cells/well on a flat-bottom 96-well plate coated with different matrices (LM511E8, LM521E8, p511, and p521), and cultured in neuronal differentiation media containing GDNF, BDNF, ascorbic acid, and dibutyryl-cAMP until day 42. For HS inhibition experiments, heparinase III (0.5 Sigma unit/mL, Sigma-Aldrich) was added to some wells before seeding the cells and incubating at 37 °C for 2 hours. On day 42, the cells were fixed with 4% paraformaldehyde and stained immunocytochemically with primary antibodies for tyrosine hydroxylase (TH) (Merck) followed by the secondary antibody (donkey anti-rabbit IgG conjugated HRP, Abcam). For high-throughput signal detection, TMB ELISA Substrate Solution and Stop Solution (Abcam) were used. The absorbance was read by an Envision 2104 microplate reader (PerkinElmer) at the 450 nm range and 620 nm as the reference. At the same time, we quantitatively evaluated live cells on day 42 using Alamar blue cell viability reagent (ThermoFisher Scientific). The medium was replaced with medium containing 10% Alamar blue, and the fluorescence intensity (excitation 560 nm, fluorescence 590 nm) was measured after incubating at 37 °C for 3 hours.
4
Modulating Histone Variants in Multiple Myeloma
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Caspase-1 inhibition was achieved with Y-VAD (50 μM) addition. Heparinase III (Sigma-aldrich) treatment (0.01 IU/mL) was used to remove HSGPG from MM cells [11 (link)]. Recombinant H2AZ (Merck-Millipore) and H4 (New England Biolabs) were added in cell culture at 2 μM or 0.5 μM depending on the experiment. Heparin (STEMCELL Technologies) was used at 20 IU/mL. DNAse I (D2) (Worthington Biochemical Corporation) was used at 100 IU/mL. Antibodies used were CD138-V421 and CD56-V450 (BD Biosciences), H2AZ, H4, H1.5, Anti-Rabbit IgG-HRP, and Anti-Rabbit IgG-Alexa Fluor-647 (Cell Signaling Technology).
5
Enzymatic Tissue Dissociation
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Heparinase III, hyaluronidase, neuraminidase and fMLP were purchased from Sigma, anesthetics were purchased from Abbeyville Veterinary Hospital, Togher, Cork.
6
Visualizing Tau Protein Internalization
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Tau monomers were conjugated to Alexa Fluor 555 (cy3) with a final degree of labeling of 3.2 (3.2 mol of dye/mole of protein) (Life Technologies, Outsourcing Facilities). Cy3-tagged oligomers were produced from cy3-tagged monomers using the Tau oligomer formation protocol described above. For internalization assays, cells were treated with 100 nM labeled tau monomers or oligomers (at 37 °C, 5% CO2) in medium. After four hours of incubation with the labeled tau species, cells were rinsed three times in warmed medium. One day later, a Hoechst 33342 stain (Life Technologies; final concentration of 1 μg/mL) was added to label nuclei in the presence of a red background suppressor (Life Technologies). Live-cell imaging was performed under a 20× magnification. High-content imaging and analysis was then used to quantify cy3-positive cells for a set threshold of cy3 fluorescence. The presence of the background suppressor dye (Life Technologies; using manufacturer’s protocol) in the media was to prevent any extracellular fluorescence40 (link). To test the effect of heparinase III (Sigma-Aldrich H8891) on tau uptake, co-cultures were treated with the 150 mU/ml enzyme for 4 h prior to the addition of cy3-tau oligomers.
7
Astrocyte Tau Uptake Dynamics
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Primary astrocyte cultures were treated with either 1 μM of Tau-Cy5 or PBS-Cy5 for between 0 to 24 h. In the case of heparin or heparinase experiments, cultures were pre-treated with 20 μg/ml of heparin (Sigma, United States) or 10 mU/ml of heparinase III (Sigma, United States) for 3 and 2 h, respectively before treatment with Tau-Cy5 or PBS-Cy5, as described above. Afterward, the cells were washed three times with PBS in order to remove excess Tau attached to the membrane. They were then prepared for immunocytochemistry or immunoblotting analysis.
8
Glycosaminoglycan Enzyme Treatment Protocol
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Heparinase I, Heparinase II, Heparinase III, and Chondroitinase ABC were purchased from Sigma Aldrich (St. Louis, MO). MEF 10.1 cells in culture were treated with Heparinase In heparinase buffer (20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 4 mM CaCl2, and 0.01% bovine serum albumin (BSA)) at a concentration of 1U/ml or chondroitinase re-suspended in chondroitinase buffer (50 mM Tris, pH 8.0, 60 mM sodium acetate and 0.02% BSA) at a concentration of 1 U/ml for 1 h at 37°C. As a control, cells were treated with enzyme buffer alone. Following incubation, the enzyme solution was removed and cells were washed with PBS to remove excess enzyme. Subsequently the cells were treated with peptide and infected with virus. Data was collected and analyzed as described above.
9
Heparinase Pretreatment Enhances AAV Transduction
10
Heparinase III Digestion of Peritoneal Cells
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