Hyaluronidase

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Hyaluronidase is an enzyme used in laboratory settings. It functions by breaking down hyaluronic acid, a component of the extracellular matrix.

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Lab products found in correlation

1 464 protocols using hyaluronidase

Sperm Capacitation and Embryo Culture Protocol

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Sperm were preincubated in a modified Krebs-Ringer bicarbonate solution (TYH) containing 0.75 mM methyl-β-cyclodextrin (MBCD, Sigma-Aldrich, St, Louis, MO, USA) that strongly promotes sperm capacitation by facilitating cholesterol efflux from the plasma membrane of the sperm [3 , 22 (link)]. Modified human tubal fluid (mHTF) with a high concentration of calcium (5.14 mM), which is bicarbonate-buffered solution, was used as the cumulus removal and fertilization mediums [23 (link), 24 (link)]. Potassium simplex optimization medium (KSOM) was used to culture embryos to the blastocyst stage [25 (link)]. Hyaluronidase was purchased from Sigma-Aldrich (400–1000 unit/mg of Hyaluronidase from bovine testes, Type I-S; Sigma-Aldrich). The titer of Hyaluronidase (Lot No. 029K7001V), measured by Sigma-Aldrich, was 801
unit/ml. A stock solution of 1% Hyaluronidase was prepared in mHTF and stored at – 20 C until use.

ISHIZUKA Y., TAKEO T., NAKAO S., YOSHIMOTO H., HIROSE Y., SAKAI Y., HORIKOSHI Y., TAKEUJI S., TSUCHIYAMA S, & NAKAGATA N. (2014). Prolonged exposure to hyaluronidase decreases the fertilization and development rates of fresh and cryopreserved mouse oocytes. The Journal of Reproduction and Development, 60(6), 454-459.

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Synovial Fluid Proteome Enrichment

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Synovial fluid sample collection was carried out as previously described [16 (link)]. The samples without detectable hemolysis were centrifuged at 400× g at 4 °C for 5 min to remove cells and debris, then aliquoted and stored at −80 °C until use.
A five hundred μL aliquoted sample was thawed and treated with 1 μg/mL hyaluronidase (Sigma-Aldrich, Gillingham, UK) at 37 °C for 1 h [16 (link)], then centrifuged at 10,000× g, at 5 °C for 10 min. The Bradford method measured the total SF protein concentration by eluding a mean of 13 μg/μL (±1.4).
The SF samples treated with 1 μg/mL hyaluronidase were concentrated by 3-kDa cut-off Amicon filter (Millipore, Billerica, MA, USA) to reach the concentration of about 25 μg/μL and treated with ProteoMiner™ Small Capacity beads (Bio-Rad Laboratories, Hercules, CA, USA). Five mg of SF’s proteome were appropriately adapted to its subcolumn and processed according to the manufacturer’s instructions. Finally, the enriched SF protein concentration was determined.

Rocchetti M.T., Bizzoca D., Moretti L., Ragni E., Moretti F.L., Vicenti G., Solarino G., Rizzello A., Petruzzella V., Palese L.L., Scacco S., Banfi G., Moretti B, & Gnoni A. (2023). A Gel-Based Proteomic Analysis Reveals Synovial α-Enolase and Fibrinogen β-Chain Dysregulation in Knee Osteoarthritis: A Controlled Trial. Journal of Personalized Medicine, 13(6), 916.

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Tumor Tissue Dissociation and Preservation

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Under sterile conditions, RCC patient tumor samples were minced into small pieces and exposed to collagenase/hyaluronidase (collagenase 1 mg/mL, hyaluronidase 0.1 mg/mL, Sigma-Aldrich) digestive solution for 120 minutes. Samples were filtered through 100 μm cell strainers, washed with PBS, and stored at −80°C.

Singh A.K., Winslow T.B., Kermany M.H., Goritz V., Heit L., Miller A., Hoffend N.C., Stein L.C., Kumaraswamy L.K., Warren G.W., Bshara W., Odunsi K., Matsuzaki J., Abrams S.I., Schwaab T, & Muhitch J.B. (2017). A Pilot Study of Stereotactic Body Radiation Therapy Combined with Cytoreductive Nephrectomy for Metastatic Renal Cell Carcinoma. Clinical cancer research : an official journal of the American Association for Cancer Research, 23(17), 5055-5065.

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Immunohistochemical Analysis of Cartilage

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Samples were prepared as previously described (Hellingman et al., 2010) . Briefly, collagen type IIstained samples were incubated for 30 min at 37 °C in 1 mg/mL pronase (Sigma-Aldrich) and 10 mg/ mL hyaluronidase (Sigma-Aldrich). 10 % normal goat serum (Southern Biotech, Uithoorn, The Netherlands) was used to block non-specific antibody binding. Sections were incubated with 0.4 mg/mL of mouse anti-collagen type II antibody (II-II/II6B3; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA). Collagen type X staining was performed by incubating samples with 0.1 % pepsin (Sigma-Aldrich) in 0.5 M acetic acid (pH 2.0), followed by 10 mg/mL hyaluronidase treatment. Rat knees were decalcified for 2 weeks in 10 % EDTA (w/v in distilled water; Sigma-Aldrich). Sections were incubated for 16 h with 1 : 10 or 1 : 100 diluted mouse anti-collagen type X antibody (X53; Quartett, Berlin, Germany; in PBS/1 % BSA). All stainings were incubated for 30 min with 2. mL Naphtol AS-MX phosphate (Sigma-Aldrich), 33 μL/mL dimethylformamide (Sigma-Aldrich) and 0.25 mg/mL levamisole (Sigma-Aldrich). Collagen type II samples were rinsed in PBS, followed by haematoxylin counterstaining. Matching mouse IgG (X0931; Dako, Fisher Scientific) isotype controls were performed for each staining.

Knuth C.A., Witte-Bouma J., Ridwan Y., Wolvius E.B, & Farrell E. (2017). Mesenchymal stem cell-mediated endochondral ossification utilising micropellets and brief chondrogenic priming. European cells & materials, 34.

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Hyaluronidase Inhibition Assay Protocol

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The hyaluronidase inhibitory assay was performed following the suggested method of Kolakul et al. [52 (link)] using 1.5 units of hyaluronidase (Sigma Aldrich) as well as hyaluronic acid solution (0.03% (w/v)) as the substrate. The precipitation of the undigested form of hyaluronic acid occurred with acid albumin solution (0.1% (w/v) BSA). Then, absorbance was measured at 600 nm using a microplate reader (BioTek ELX800; BioTek Instruments, Colmar, France). hyaluronidase’s inhibitory effect was expressed as the percent of inhibition relative to the control (extraction solvent) for every extract sample. Oleanolic acid (10 µM) was used as a specific inhibitor of hyaluronidase.

Tungmunnithum D., Drouet S, & Hano C. (2022). Validation of a High-Performance Liquid Chromatography with Photodiode Array Detection Method for the Separation and Quantification of Antioxidant and Skin Anti-Aging Flavonoids from Nelumbo nucifera Gaertn. Stamen Extract. Molecules, 27(3), 1102.

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Isolation of Mammary Gland Cells

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E14 Lgr5-GFP embryos were collected and the whole skin containing the MGs was dissected. Tissues were digested in 300U/ml collagenase (Sigma cat#C0130) + 300μg/ml hyaluronidase (Sigma cat#4272) diluted in HBSS for 1h30 at 37°C under shaking. EDTA at a final concentration of 5mM was added for 3min, followed by two washes in 10% FBS/PBS and 2% FBS/PBS before filtration through a 40μm mesh. Adult MGs were dissected and the lymph nodes removed. Tissues were briefly washed in HBSS, and chopped in 1mm³ pieces. Chopped tissues were digested in HBSS + 300 U/ml collagenase (Sigma cat#C0130) + 300μg/ml hyaluronidase (Sigma cat#4272) for 2h at 37°C under agitation. Physical dissociation using a P1000 pipette was done every 15mins throughout the enzymatic digestion. EDTA 5mM was added for 5 minutes, followed by 0,25% Trypsin-EGTA for 1 min before filtration through a 70-μm mesh, and 2 successive washes in 2% FBS/PBS.

Wuidart A., Sifrim A., Fioramonti M., Matsumura S., Brisebarre A., Brown D., Centonze A., Dannau A., Dubois C., Van Keymeulen A., Voet T, & Blanpain C. (2018). Early lineage segregation of multipotent embryonic mammary gland progenitors. Nature cell biology, 20(6), 666-676.

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Macrophage Depletion and Adhesion Molecule Blocking for Intestinal Injury

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Peritoneal macrophages were depleted by intraperitoneal administration of 100 μL/mouse (0.69 mol/L) clodronate liposome (#CP-020-020; clodronateliposomes.org, Vrije Universiteit, Netherlands) 4 days prior to the experiment. The same dose of PBS-liposome (#CP-020-020) was used for the control experiment. For adhesion molecule blocking experiments, mice were administered either 50 μg anti-CD44 (#CL8944AP; KM81, Cedarlane) monoclonal antibody or isotype control antibody 1 h prior to the injury. Apyrase treatment was performed by intraperitoneal administration of 25 U Apyrase (#A2230; Sigma-Aldrich). For blocking ATP receptor, mice received 10 μg P2X7 antagonist (#A438079; R&D) intraperitoneally 1 h prior to the injury. Hyaluronidase treatment was performed by intraperitoneal administration of 100 U Hyaluronidase (#H3506; Sigma-Aldrich) just after the intestinal injury. Each reagent was diluted in PBS as appropriate.

Honda M., Kadohisa M., Yoshii D., Komohara Y, & Hibi T. (2021). Directly recruited GATA6 + peritoneal cavity macrophages contribute to the repair of intestinal serosal injury. Nature Communications, 12, 7294.

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Hyaluronidase Inhibition by ZnO-NPs

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A combination of hyaluronic acid and hyaluronidase (1.5 units; Sigma Aldrich) was mixed to create a substrate solution to test ZnO-NPs for their inhibitory ability against hyaluronidase. Undigested hyaluronic acid was precipitated with a BSA-containing acid albumin solution (0.1 percent w/v). A microplate reader measured the absorbance at 600 nm. It was measured in terms of the percentage of inhibition of NPs in comparison to the control.

Rehman H., Ali W., Zaman Khan N., Aasim M., Khan T, & Ali Khan A. (2022). Delphinium uncinatum mediated biosynthesis of zinc oxide nanoparticles and in-vitro evaluation of their antioxidant, cytotoxic, antimicrobial, anti-diabetic, anti-inflammatory, and anti-aging activities. Saudi Journal of Biological Sciences, 30(1), 103485.

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Isolation and IVF of Mouse Oocytes

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MII oocytes were collected from 6‐ to 10‐week‐old C57BL/6NHsd mice. Mice were treated with pregnant mare serum gonadotropin (5 IU, PMSG; Sigma, St. Louis, MO) and human chorionic gonadotropin (5 IU, HCG; Sigma). Female mice were manually restrained for intraperitoneal (IP) injections. HCG was IP injected into female mice 47‐49 h after their last PMSG injection. Approximately 16 h after HCG injection, oviducts were collected from female mice and placed in M2 media (Sigma). Each oviduct was then transferred to a dish containing M2 media and hyaluronidase (Sigma). The ampulla was incised and washed with M2 media to obtain the oocytes. Oocytes were collected and placed into a 100‐µL drop of KSOM (Sigma) using embryo‐tested mineral oil (Sigma) that had been equilibrated at 37°C in a 5% CO₂ atmosphere. When required, cumulus cells were treated with bovine testes hyaluronidase (0.1%, Sigma). Mouse sperm was harvested from the cauda epididymis of 10‐15‐week‐old C57BL/6NHsd male mice, and the spermatozoa were suspended in M2 medium for 20 min. All animal experiments and procedures were approved by the Institutional Animal Care and Use Committee of Sun Yat‐Sen Memorial Hospital, Sun Yat‐Sen University.

Zhang Z., Wang T., Huang J., Huang Y, & Zhang Q. (2020). Microinjection manipulation decreases the expression of GABA‐A receptor signaling pathway genes in mouse embryos derived using intracytoplasmic sperm injection. Journal of Clinical Laboratory Analysis, 35(1), e23584.

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Hyaluronidase and 4-MU treatment on EVs uptake

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To remove HA matrix before seeding in 24-well plates, after trypsin detachment, both ACHs and FLSs were split in two, and one aliquot was treated for 10 min at 37 °C with 10 U/mL hyaluronidase (Sigma-Aldrich), whereas the other aliquot was incubated for the same time without the enzyme (mock). After two washes, treated cells were supplemented with 1 mM 4-methylumbelliferone (4-MU) (Sigma-Aldrich), an HA synthesis inhibitor, in the complete medium, and eventually, both hyaluronidase + 4-MU treated and untreated cells were seeded as previously described. 4-MU was left all night in treated cells. The following day, CFSE-labeled EVs (ratio of 50,000 EVs per seeded cell) were added in both treated and untreated cells and 4-MU was maintained in treated cells. Flow cytometry was performed at 0 (before EV addition) and 24 h and 4-MU treated (±EVs) vs untreated (±EVs) samples compared. Fluorescence values were normalized by detected events under SSC-A vs FSC-A plot.

Ragni E., Palombella S., Lopa S., Talò G., Perucca Orfei C., De Luca P., Moretti M, & de Girolamo L. (2020). Innovative Visualization and Quantification of Extracellular Vesicles Interaction with and Incorporation in Target Cells in 3D Microenvironments. Cells, 9(5), 1180.

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