Collagenase d from clostridium histolyticum

Manufactured by Roche

Sourced in Ireland

Collagenase D from Clostridium histolyticum is an enzyme used for the dissociation of various tissue types. It is a crude collagenase preparation with a high broad-spectrum collagenolytic activity.

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4 protocols using collagenase d from clostridium histolyticum

Optimized Cell Culture Techniques for In Vitro Studies

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Dulbecco's modified Eagle medium (DMEM), MEM non-essential amino acids and 0.25% trypsin/EDTA solution were purchased from Invitrogen (Gaithersburg, MD). Platelet-derived growth factor BB isotype (PDGF), GM6001 (also known as galardin or ilomastat) were obtained from Millipore (Billerica, MA). BB-94 (also known as batimastat) was purchased from Tocris (Bristol, UK). Fetal bovine serum (FBS), fatty acid-free and fraction V bovine serum albumin (BSA), RPMI vitamin mix, HEPES, DMSO, thymidine and Sodium bicarbonate were obtained from Sigma-Aldrich (St. Louis, MO). Penicillin, streptomycin, and amphotericin B were obtained from Lonza inc. (Walkersville, MD). Type I rat tail collagen was purchased from BD Biosciences (Bedford, MA). PureCol® Type I bovine collagen was purchased from Advanced BioMatrix, Inc. (San Diego, CA). Alexa Fluor 488 and Propidium Iodine (PI) were obtained from Molecular Probes, Inc. (Eugene, OR). Collagenase D from Clostridium histolyticum and RNase (DNase free) were purchased from Roche (Indianapolis, IN). Rabbit eyes were purchased from Pel Freez (Rogers, AR). Human glu-plasminogen was purchased from Haematologic Tech (Essex Junction, VT). Glass bottom dishes were purchased from MatTek (Ashland, MA). MT1-MMP mouse-anti-human monoclonal antibody (C9, Santa Cruz, TX)

Zhou C, & Petroll W.M. (2014). MMP Regulation of Corneal Keratocyte Motility and Mechanics in 3-D Collagen Matrices. Experimental eye research, 121, 147-160.

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Isolation and Culture of Adipose-Derived Stromal Vascular Fraction

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The stromal vascular fraction was isolated from adipose tissue and prepared for flow cytometry or culture. Briefly, the adipose tissue deposits (epididymal and inguinal white adipose tissue; E-WAT and I-WAT, respectively) were removed, weighed, minced and digested with 1 mg/ml Collagenase D from Clostridium histolyticum (Roche, Dublin, Ireland) for 30 min at 37°C for 30 min with shaking. Digested tissue was passed through a 70μM cell strainer and cells pelleted at 1,500 rpm for 5 min. Red blood cell contamination was removed by incubation with a hypotonic lysis solution. Cells were used for flow cytometry or cultured for 2 h in RPMI-1640 supplemented with 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin at a density of 2 × 10⁶ cells/ml at 37°C with 5% CO₂. Gene expression was assessed in adherent cells.

Hams E., Roberts J., Bermingham R., Hogan A.E., O'Shea D., O'Neill L, & Fallon P.G. (2020). Role for Retinoic Acid-Related Orphan Receptor Alpha (RORα) Expressing Macrophages in Diet-Induced Obesity. Frontiers in Immunology, 11, 1966.

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Protein Release from 3D-Printed Scaffolds

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Protein absorption and release study was carried out as previously reported [9 (link)]. Model proteins lysozyme from chicken egg (Sigma-Aldrich, UK) and bovine serum albumin (BSA, Sigma-Aldrich, UK) were solubilized in HBSS (Thermo-Fisher, UK) at 10 µg/mL and 100 µg/mL, respectively. To investigate the effect of the nanoclay particles on drug release, 3D scaffolds were printed using nanocomposite (LAB) and Laponite-free controls (alginate-bone-ECM (AB)) to allow the absorption of the compounds of interest after ionic crosslinking. The 3D-printed constructs (n=3) were soaked in lysozyme or BSA for 1 h, and their release was monitored over 24 h. BSA and lysozyme were quantified with a RAPID kit (Sigma-Aldrich, UK) using a GloMax Discover microplate reader (Promega). The supernatant was collected after 1, 2, 4, 8, 10, 20, and 24 h following adsorption. Collagenase D (from Clostridium histolyticum, Roche Diagnostics GmbH) was added 24 h after adsorption to stimulate material degradation and cargo release. BSA and lysozyme release was quantified after 1, 2, 4, 8, 10, 20, and 24 h.

Kim Y.H., Kanczler J.M., Lanham S., Rawlings A., Roldo M., Tozzi G., Dawson J.I., Cidonio G, & Oreffo R.O. (2024). Biofabrication of nanocomposite-based scaffolds containing human bone extracellular matrix for the differentiation of skeletal stem and progenitor cells. Bio-Design and Manufacturing, 7(2), 121-136.

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Isolation and Phenotyping of Murine Ocular Immune Cells

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Eyes were harvested and pooled for each experimental group. After removal of lens, eyes were minced and digested with 1 mg/ml of collagenase D from Clostridium histolyticum (Roche Diagnostics, Mannheim, Germany) in culture medium consisting of RPMI supplemented with 10% fetal calf serum, 1% penicillin/streptomycin, 2% L-glutamine, and 5 × 10⁻⁵ M of 2-ME for 30 minutes at 37°C. Cells were extracted through a 40 μm cell strainer, live cells were counted using a hemocytometer and trypan dye exclusion, washed in flow cytometry buffer (0.2% BSA, 0.1% sodium azide in PBS) and stained with fluorochrome-conjugated antibodies against mouse CD45.2 (Clone 104), CD3 (17A2), CD4 (GK1.5), CD8 (53–6.7), CD19 (6D5), CD11b (M1/70), CD11c (N418), I-Ab (AF6–120.1), and Ly6C (HK1.4). Antibodies were purchased from Biolegend. Flow cytometry was performed on an LSR II cytometer in the Molecular Biology Core Facility of the Kellogg Eye Center and data were analyzed using FlowJo v10.0.7 software (TreeStar, Inc.). Cells were gated for lymphocytes (low side scatter and intermediate forward scatter) and then live singlet cells that are positive for CD45.2 marker. For calculation of total number of cells, equal number of cells in lymphocyte gate were considered.

Nikoopour E., Lin C.M., Sheskey S., Heckenlively J.R, & Lundy S.K. (2019). Immune Cell Infiltration into the Eye is Controlled by Interleukin 10 in Recoverin-Induced Autoimmune Retinopathy. Journal of immunology (Baltimore, Md. : 1950), 202(4), 1057-1068.

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