Hyaluronidase type 1 s

Manufactured by Merck Group

Sourced in United States, Germany

Hyaluronidase type I-s is a laboratory enzyme used to break down hyaluronic acid, a naturally occurring polysaccharide in the body. It is commonly used in research applications to facilitate the movement and distribution of other injected substances.

Automatically generated - may contain errors

Lab products found in correlation

Collagenase type xi, by Merck Group (15 mentions) Collagenase type 1, by Merck Group (13 mentions) Dnase 1, by Merck Group (12 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (9 mentions) Dnase, by Merck Group (7 mentions) Dnase 1, by Roche (5 mentions) Gentamicin, by Thermo Fisher Scientific (4 mentions) Collagenase type ia, by Merck Group (3 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions) Amphotericin b, by Merck Group (3 mentions) Collagenase type 1, by Thermo Fisher Scientific (3 mentions) Collagenase type 1, by Worthington (3 mentions) Collagenase type 4, by Merck Group (2 mentions) Glutamine, by Merck Group (2 mentions) Cell strainer, by BD (2 mentions) Cd11c pe, by BioLegend (2 mentions) Fc blocking antibody, by Thermo Fisher Scientific (2 mentions) Cd19 apc cy7, by BioLegend (2 mentions) Cd3 apc, by BioLegend (2 mentions) Cd45 fitc, by BioLegend (2 mentions)

43 protocols using hyaluronidase type 1 s

Oocyte Fertilization and Embryo Transfer

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Cumulus-intact oocytes were collected in 0.2 ml of TYH medium and inseminated with capacitated sperm (final concentration, 100 sperm/μl). After 2 h of incubation at 37 °C under 5% CO₂ in air, cumulus cells were dispersed by brief treatment with hyaluronidase (Type-IS, 150 units/ml, Sigma). The embryos immediately after fertilisation were injected with the mixture of histone H2B mRNA and γH2A.X Fab fragment as described above and further cultured in KSOM. Two-cell stage embryos were transferred into the oviduct of surrogate mothers (ICR strain) on Day 1 of pseudo-pregnancy following mating with vasectomised ICR males.

Yamagata K., Nagai K., Miyamoto H., Anzai M., Kato H., Miyamoto K., Kurosaka S., Azuma R., Kolodeznikov I.I., Protopopov A.V., Plotnikov V.V., Kobayashi H., Kawahara-Miki R., Kono T., Uchida M., Shibata Y., Handa T., Kimura H., Hosoi Y., Mitani T., Matsumoto K, & Iritani A. (2019). Signs of biological activities of 28,000-year-old mammoth nuclei in mouse oocytes visualized by live-cell imaging. Scientific Reports, 9, 4050.

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Aorta Single-Cell Isolation Protocol

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Aortas were dissected from the aortic root to the top of the diaphragm after perfusion of the vasculature, and cleared of adventitial fat. Single cell suspensions were obtained by digesting the tissue for 1 h at 37°C in PBS with 450 U/ml collagenase type I, 125 U/ml collagenase Type XI, 60 U/ml hyaluronidase type I-S, and 60 U/ml DNase I (all from Sigma), as previously described [28] (link). Digests were filtered thru a 70 µm filter and resuspended in FACS buffer.

Vu D.M., Tai A., Tatro J.B., Karas R.H., Huber B.T, & Beasley D. (2014). γδT Cells Are Prevalent in the Proximal Aorta and Drive Nascent Atherosclerotic Lesion Progression and Neutrophilia in Hypercholesterolemic Mice. PLoS ONE, 9(10), e109416.

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Isolation and Immortalization of Mammary Breast Cancer Cells

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End-stage tumors of Ctsl^fl/fl mice were carefully harvested and disrupted by enzymatic dissociation with DNAse I (#DN25, Sigma-Aldrich), Hyaluronidase Type I-S (#H3506, Sigma-Aldrich) and Collagenase Type IV (#C5138, Sigma-Aldrich). Cells were kept as monolayer cultures in a cell culture incubator under sterile conditions at 37 °C, 5% CO₂, and 91.0% rH. Once spontaneously immortalized after 10–12 passages, mammary breast cancer cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco/Invitrogen, Paisley, UK) supplemented with 1% penicillin/streptomycin, 1% L-glutamine (both from Gibco/Invitrogen), and 10% fetal calf serum (PAN-Biotech, Aidenbach, Germany), in humidified air containing 5% CO₂. Cells were also kept under low-serum conditions (1%). Cells were transduced with a doxycycline-inducible retroviral Cre-construct (kindly provided by Tilman Brummer). Induction by 2 µg/mL doxycycline took place every 48 h for 6 days. In the indicated experiments, 10 μM of the pan cysteine Cathepsin inhibitor E64d were supplemented to the full medium of Ctsl^fl/fl cells every 48h for 4 days prior to lysis.

Parigiani M.A., Ketscher A., Timme S., Bronsert P., Schlimpert M., Kammerer B., Jacquel A., Chaintreuil P, & Reinheckel T. (2020). Conditional Gene Targeting Reveals Cell Type-Specific Roles of the Lysosomal Protease Cathepsin L in Mammary Tumor Progression. Cancers, 12(8), 2004.

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HNPGL Primary Cell Culture Protocol

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The primary culture of HNPGL was performed according to protocols previously described [23] (link). Briefly, tumor tissues were cut into 1 mm³ segments, washed with phosphate-buffered saline (PBS), and then incubated in media with enzyme mixture containing 250 U/mL Hyaluronidase Type I-S (#C1639, Sigma-Aldrich) and 160 U/mL Collagenase Type I (#3506, Sigma-Aldrich) in DMEM/F12 (#11330032, Gibco) for 18 h at 37 °C. After the enzymatic incubation of the culture, the cells were re-suspended in DMEM/F12 medium with 10% fetal bovine serum (FBS) (#1414426, Gibco), passed through a 200 mesh filter screen, and finally transferred to 6 cm culture dishes pre-coated with poly-Lornithine and laminin (#354232, BD Biosciences).

Wang Z., Chen H., Xue L., He W., Shu W., Wu H, & Wang Z. (2021). High throughput proteomic and metabolic profiling identified target correction of metabolic abnormalities as a novel therapeutic approach in head and neck paraganglioma. Translational Oncology, 14(8), 101146.

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Isolation and Culture of Fibroblast-Like Synoviocytes

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FLS were obtained as previously described [7 (link)]. Briefly, freshly obtained synovial tissues were minced and incubated with a solution containing DNase (0.15mg/ml), hyaluronidase type I-S (0.15 mg/ml), and collagenase type IA (1 mg/ml) (Sigma, St.Louis, MO) in DMEM (Invitrogen, Carlsbad, CA) for 1 hour at 37°C. Cells were washed and re-suspended in complete media containing DMEM supplemented with 10% FBS (Invitrogen), glutamine (300 ng/ml), amphotericin B (250 μg/ml) (Sigma), and gentamicin (20 ng/ml) (Invitrogen). After overnight culture, non-adherent cells were removed and adherent cells cultured. All experiments were performed with FLS after passage four (>95% FLS purity).

Laragione T., Cheng K.F., Tanner M.R., He M., Beeton C., Al-Abed Y, & Gulko P.S. (2015). THE CATION CHANNEL TRPV2 IS A NEW SUPPRESSOR OF ARTHRITIS SEVERITY, JOINT DAMAGE AND SYNOVIAL FIBROBLAST INVASION. Clinical immunology (Orlando, Fla.), 158(2), 183-192.

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Immunohistochemical Analysis of Paraffin Sections

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Immunohistochemical analysis of paraffin sections was performed using the ChemMate EnVision system (Dako) according to the manufacturer's instructions. Deparaffinized and rehydrated sections were immersed in 0.3% hydrogen peroxide in methanol for 30 min at room temperature to block endogenous peroxidase activities. For SOX9, K17 and Ki-67 immunostaining, antigen retrieval was performed by first autoclaving sections in citric acid buffer (pH 6.0) at 121°C for 10 min. To analyze the stromal expression of perlecan, the sections were incubated with hyaluronidase (Type IS, 330 UI/ml; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 30 min prior to immunostaining. After antigen retrieval, the sections were incubated with 5% milk protein in phosphate-buffered saline (PBS) for 30 min and then with the primary antibodies overnight at 4°C. After washing with PBS, the sections were reacted with EnVision (Dako) for 60 min at room temperature. Peroxidase reaction products were developed with 3,3′-diaminobenzidine, and the sections were counterstained with hematoxylin. In the control experiments, the primary antibodies were replaced with the appropriate negative control immunoglobulin (cat. no. X0931 for mouse IgG1 and cat. no. X0903 for rabbit Ig fraction; Dako).

Sumita Y., Yamazaki M., Maruyama S., Abé T., Cheng J., Takagi R, & Tanuma J.I. (2018). Cytoplasmic expression of SOX9 as a poor prognostic factor for oral squamous cell carcinoma. Oncology Reports, 40(5), 2487-2496.

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Isolation and Characterization of Aorta Cells

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Single aorta cells were prepared using methods described below with modification. The isolated aorta was dissected and cut into small pieces. Aorta pieces were digested with 125 U/ml collagenase type XI, 60 U/ml hyaluronidase type I-s, 60 U/ml DNase I, and 450 U/ml collagenase type I purchased from Sigma-Aldrich in PBS containing 20 mM HEPES at 37°C for 1 hour. Cells were incubated with propidium iodide (catalog 5135, R&D System) and stained with CD45-Pacific Blue (catalog 57-0451) and CD14 APC at 1:1,000 (catalog 17-0141) (purchased from Bioscience) and CD19 Alex Fluor 488 at 1: 1,000 (catalog 302219), TCR PerCP/Cys5.5 at 1:1,000 (catalog 109227), CD11c PE/Cy7 at 1:1,000 (catalog 117317), and CD68 PE at 1:1,000 (catalog 12-0681) (purchased from BioLegend). Data analysis was performed using FlowJo software.

Park K., Mima A., Li Q., Rask-Madsen C., He P., Mizutani K., Katagiri S., Maeda Y., Wu I.H., Khamaisi M., Preil S.R., Maddaloni E., Sørensen D., Rasmussen L.M., Huang P.L, & King G.L. (2016). Insulin decreases atherosclerosis by inducing endothelin receptor B expression. JCI Insight, 1(6), e86574.

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Single-Cell Isolation and Characterization

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Cells were isolated on ice, and single‐cell suspensions were prepared for use.^[⁴⁹^] To obtain single cells from an aorta, the whole aortas were minced into small pieces and then digested in 1X Aorta Dissociation Enzyme stock Solution (125 U mL⁻¹ collagenase type XI, 60 U mL⁻¹ hyaluronidase type I‐s, 60 U mL⁻¹ DNase I, and 450 U mL⁻¹ collagenase type I, all enzymes were obtained from Sigma‐Aldrich) at 37°C for 1 h. Cell suspensions were prepared by filtering them through a cell strainer (#322 350, 70 µm size, BD Bioscience, Billerica, MA). Cells were then stained by incubation with surface markers for 30 min on ice. For intracellular staining, cells were fixed and permeabilized using a Intracellular Fixation and Permeabilization buffer set (No. 00‐5523‐00, eBioscience, San Diego, CA) after surface marker staining, followed by staining with antibodies against intracellular antigens. For cell apoptosis detection, SMCs were collected, washed with PBS twice and stained with the annexin V and 7‐AAD according to the manufacturer's instructions. Stained cells were analyzed with a flow cytometer (FACS Aria III, BD). All reagents used for flow cytometry are listed in Table S1 (Supporting Information).

Li J., Xia N., Li D., Wen S., Qian S., Lu Y., Gu M., Tang T., Jiao J., Lv B., Nie S., Hu D., Liao Y., Yang X., Shi G, & Cheng X. (2022). Aorta Regulatory T Cells with a Tissue‐Specific Phenotype and Function Promote Tissue Repair through Tff1 in Abdominal Aortic Aneurysms. Advanced Science, 9(9), 2104338.

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Dissociation and Flow Cytometry of Aortic Cells

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Mouse abdominal aortas were harvested, minced, and digested in enzymatic digestion solution containing 450 U/mL collagenase type I (Gibco, #17100–017), 125 U/mL collagenase type XI (Sigma-Aldrich, #C7657), 60 U/mL hyaluronidase type I-s (Sigma-Aldrich, #H3506) and 60 U/mL DNase-I (Roche, #10104159001) at 37°C for 60 minutes. Single cells were released using a 70μm cell strainer and stained with the indicated antibodies, followed by flow cytometry analysis on a MoFlo Astrios cell sorter (Beckman Coulter, Brea, CA). Antibodies used for flow cytometry are listed in the major resource table. Dot plots were generated using the FlowJo Software.

Chang Z., Zhao G., Zhao Y., Lu H., Xiong W., Liang W., Sun J., Wang H., Zhu T., Rom O., Guo Y., Fan Y., Chang L., Yang B., Garcia-Barrio M.T., Lin J.D., Chen Y.E, & Zhang J. (2020). BAF60a Deficiency in Vascular Smooth Muscle Cells Prevents Abdominal Aortic Aneurysm by Reducing Inflammation and Extracellular Matrix Degradation. Arteriosclerosis, thrombosis, and vascular biology, 40(10), 2494-2507.

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Single-cell Isolation and Analysis of Mouse Aorta

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Single-cell suspensions were prepared from the mouse aortas as described^{20 (link)}. Briefly, mice were anesthetized and perfused through the heart with PBS containing 10 U ml⁻¹ heparin to remove blood from all vessels. After careful removal of the perivascular adipose tissue, the aortas were minced into small pieces and digested with 125 U ml⁻¹ collagenase type XI, 60 U ml⁻¹ hyaluronidase type I-s, 60 U ml⁻¹ DNase1, and 450 U ml⁻¹ collagenase type I (all enzymes were obtained from Sigma-Aldrich) in PBS containing 20 mM Hepes at 37°C for 3 hrs. For separating adventitia and media, the aortas were predigested in enzyme cocktail (300U ml⁻¹ Collagenase I and 10U ml⁻¹ Elastase in PBS) at 37°C for 15 minutes and then the adventitia was carefully peeled away from the aorta. After filtering through a 70 μm filter, cells were resuspended in FACS buffer, and incubated with Fc-blocking antibody (eBioscience) for 15 min on ice before being stained with specific antibodies. Antibodies used were as follows: FITC-CD45.2, PE-CD11c, APC-CD3, APC-Cy7-CD19, PE-Cy7-NK1.1, PerCy5.5-CD11b (all antibodies were obtained from Biolegend and used at 1:100 dilution). Cells were also stained with propidium iodide. After washing, immunofluorescence was detected by an LSR II (BD Biosciences). Data were analyzed using FlowJo (Tree Star Inc.) software.

He C., Medley S.C., Hu T., Hinsdale M.E., Lupu F., Virmani R, & Olson L.E. (2015). PDGFRβ signaling regulates local inflammation and synergizes with hypercholesterolemia to promote atherosclerosis. Nature communications, 6, 7770.

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