Collagenase from clostridium histolyticum type 4

Manufactured by Merck Group

Sourced in United States, Germany

Collagenase from Clostridium histolyticum type IV is an enzyme that hydrolyzes collagen, the main structural protein in various connective tissues. It is used for tissue dissociation and cell isolation applications.

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Lab products found in correlation

Sodium pentobarbital, by Sanofi (3 mentions) 2 2 dinitro 5 5 dithiodibenzoic acid dtnb, by Merck Group (3 mentions) Lactate dehydrogenase ldh kit, by Randox (3 mentions) D glucose, by Merck Group (3 mentions) Sodium chloride (nacl), by Merck Group (3 mentions) Potassium chloride (kcl), by Merck Group (3 mentions) Nahco3, by Merck Group (3 mentions) Ethylene glycol tetraacetic acid (egta), by Merck Group (3 mentions) Cacl2 2h2o, by Merck Group (2 mentions) Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (2 mentions) 2 thiobarbituric acid, by Merck Group (2 mentions) Nylon cell strainer, by BD (2 mentions) Percoll, by GE Healthcare (2 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions) Anti mouse cd16 32, by BD (1 mentions) Trypsin inhibitor type ii s, by Merck Group (1 mentions) Dnase 1, by Merck Group (1 mentions) N 2 hydroxyethylpiperazine n 2 ethanesulfonic acid hepes, by Merck Group (1 mentions) Mgcl2 6h2o, by Merck Group (1 mentions) Percoll, by Merck Group (1 mentions)

18 protocols using collagenase from clostridium histolyticum type 4

Cytokine and Signaling Analysis in Infected Lung

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For cellular analysis, inflammatory cytokines measurement and signaling pathway analysis, further experimental infections as described above were performed, but the mice only received 100 kU/kg dosage of rhCAT.
On days 2 and 4 post-infection, the mice (n = 4, per group) were euthanized. Lungs were harvested from PBS-perfused mice and diced using surgical scissors. Diced tissue was suspended in 4 mL of CDTI buffer [0.5 mg/mL collagenase from Clostridium histolyticum type IV (Sigma), 50 U/mL Dnase I (Sigma), 1 mg/mL trypsin inhibitor type Ii-s (Sigma) in DMEM] for 1 h at 37 °C. The suspension was then passed through a 100-μm filter, and unwanted red blood cells were lysed using red blood cell lysis buffer [0.02 Tris–HCl (pH 7.4), 0.14 NH₄Cl]. Inflammatory cells were purified by centrifugation in 35 % PBS-buffered Percoll (GE Healthcare Life Sciences) at 500 × g for 15 min. Cell pellets were resuspended in staining buffer (RPMI-1640 medium), and Fc receptors were blocked using 25-μg/mL anti-mouse CD16/32 (BD Biosciences). Cells were stained with fluorescently labeled antibodies against the following mouse proteins: CD11b⁺, F480⁻, Ly6G⁺ (Neutrophils), CD11b⁺, F480⁺, and Ly6G⁻ (Macrophage/monocytes).

Shi X., Shi Z., Huang H., Zhu H., Zhou P., Zhu H, & Ju D. (2014). Ability of Recombinant Human Catalase to Suppress Inflammation of the Murine Lung Induced by Influenza A. Inflammation, 37(3), 809-817.

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Neuronal Cell Isolation and Characterization

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In our experiments, pentobarbital sodium (Sanofi, France), N-(2-hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid) (HEPES) (Sigma Aldrich, Germany), NaCl (Merck, Germany), KCl (Merck), D-glucose (Merck), NaHCO₃ (Merck), KH₂PO₄ (Scharlau Chemie SA, Spain), CaCl₂·2H₂O (Merck), MgSO₄·7H₂O (Fluka AG, Germany), collagenase from Clostridium histolyticum type IV (Sigma Aldrich), albumin, bovine serum fraction V, minimum 98% (Sigma Aldrich), ethylene glycol tetraacetic acid (Sigma Aldrich), 2-thiobarbituric acid (TBA) (4,6-dihydroxypyrimidine-2-thiol) (Sigma Aldrich), trichloroacetic acid (TCA) (Valerus, Bulgaria), 6-hydroxydopamine (Merck), 2,2′-dinitro-5,5′-dithiodibenzoic acid (DTNB) (Merck), lactate dehydrogenase (LDH) kit (Randox, UK), D(+) sucrose (Fluka, Germany), NaH₂PO₄ (Merck), MgCl₂·6H₂O, Percoll (Sigma Aldrich), (3-[4,5-dimethylthiazol -2-yl]-2, 5diphenyl-tetrazolium bromide) (Sigma Aldrich), dimethyl sulfoxide (DMSO) (Valerus, Bulgaria) were used.

Kondeva-Burdina M., Krasteva I, & Mitcheva M. (2014). Effects of rhamnocitrin 4-β-D-galactopyranoside, isolated from Astragalus hamosus on toxicity models in vitro. Pharmacognosy Magazine, 10(Suppl 3), S487-S493.

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Isolation of Mouse Primary Hepatocytes

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Mouse primary hepatocytes were isolated as described36 (link)37 (link). Mice were anaesthetized by intraperitoneal injection of 50 mg/kg pentobarbital. The portal vein was cannulated with a 23-gauge plastic cannula. Mouse livers were perfused with Hank’s Balanced Salt Solution (HBSS, Cat # 14170-112, Thermo Fisher Scientific) containing 0.19 g/L EDTA. Simultaneously, the inferior vena cava was cut open. Subsequently, livers were perfused with HBSS, calcium, magnesium buffer (Cat # 14025092, Thermo Fisher Scientific) with 0.8 mg/mL Collagenase from Clostridium histolyticum type IV (Sigma, St. Louis, MO). Primary hepatocytes were released and collected in a 50 mL centrifuge tube. After centrifugation at 50 × g for 5 minutes and washing with DMEM, cells were cultured in DMEM/10%FBS (Atlanta Biologicals, Georgia, USA) in 6-well plates pre-coated with 0.1% gelatin (Sigma-Aldrich).

Xu J., Xu Y., Li Y., Jadhav K., You M., Yin L, & Zhang Y. (2016). Carboxylesterase 1 Is Regulated by Hepatocyte Nuclear Factor 4α and Protects Against Alcohol- and MCD diet-induced Liver Injury. Scientific Reports, 6, 24277.

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Isolation and Culture of Primary Mouse Hepatocytes

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Mouse primary hepatocytes were isolated as described.^[¹¹^] Mice were anesthetized by intraperitoneal injection of Xylazine/Ketamine. The portal vein was cannulated with a 23‐gauge plastic cannula. Mouse livers were perfused with Hank's Balanced Salt Solution (HBSS; 14170112; Thermo Fisher Scientific). Subsequently, livers were perfused with HBSS with calcium and magnesium (14,025,092; Thermo Fisher Scientific) containing 0.8 mg/ml collagenase from Clostridium histolyticum type IV (Sigma). Primary hepatocytes were released and collected in a 50‐ml centrifuge tube. After centrifugation at 50 g for 3 min and washing with Dulbecco's modified Eagle's medium (DMEM), cells were cultured in DMEM plus 10% fetal bovine serum (FBS) in plates or 6‐well dishes precoated with 0.1% gelatin. Primary hepatocytes were cultured in DMEM containing vehicle or lipid mixture (100 μm palmitate, 100 μm oleic acid, 100 μm linoleic acid, and 1 μg/ml cholesterol). After 24 or 48 h, messenger RNA (mRNA) levels and intracellular lipid levels were determined.

Cassim Bawa F.N., Xu Y., Gopoju R., Plonski N., Shiyab A., Hu S., Chen S., Zhu Y., Jadhav K., Kasumov T, & Zhang Y. (2022). Hepatic retinoic acid receptor alpha mediates all‐trans retinoic acid's effect on diet‐induced hepatosteatosis. Hepatology Communications, 6(10), 2665-2675.

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Isolation and Culture of Mouse Fibroblast-Like Synoviocytes

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Mouse FLS were cultured as described previously [12 (link)]. In brief, mice were killed, and their skin was removed from the hind paws, which were then cut 1 mm above the ankle joint and placed into Hanks’ balanced salt solution. After a wash, the paws were dissected between the toe and ankle joints using a scalpel blade, and the dissected tissue was placed into DMEM (containing 1% penicillin-streptomycin and 10% FBS) with 10 mg/ml collagenase (from Clostridium histolyticum type IV; Sigma-Aldrich, Gillingham, UK) and placed into a shaking incubator for 1.5 h at 37 °C. Cells (and some tissue debris) were pelleted by centrifugation, re-suspended in DMEM and plated out. Cells were cultured in DMEM to passage 3 before further experimental use, in order to obtain a purified population of FLS.

Hand L.E., Dickson S.H., Freemont A.J., Ray D.W, & Gibbs J.E. (2019). The circadian regulator Bmal1 in joint mesenchymal cells regulates both joint development and inflammatory arthritis. Arthritis Research & Therapy, 21, 5.

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Liver Oxidative Stress Evaluation

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The chemicals used in the experiments were: Pentobarbital sodium (Sanofi, France), (N-[2-Hydroxyethyl]piperazine-N’-[ethanesulfonic acid]) (HEPES) (Sigma-Aldrich, Germany), NaCl (Merck, Germany), KCl (Merck), D-glucose (Merck), NaHCO₃ (Merck), KH₂PO₄ (Scharlau Chemie SA, Spain), CaCl₂.2H₂ O (Merck), MgSO₄.7H₂ O (Fluka AG, Germany), collagenase from Clostridium histolyticum type IV (Sigma-Aldrich), albumin, bovine serum fraction V, minimum 98% (Sigma-Aldrich), EGTA (Sigma-Aldrich), 2-thiobarbituric acid (4,6-dihydroxypyrimidine-2-thiol; TBA) (Sigma-Aldrich), trichloroacetic acid (TCA) (Valerus, Bulgaria), 2,2’- dinitro-5,5’- dithiodibenzoic acid (DTNB) (Merck), lactate dehydrogenase (LDH) kit (Randox, UK), t-BuOOH (Sigma-Aldrich), and CCl₄ (Merck).

Kondeva-Burdina M., Valcheva-Kuzmanova S., Markova T., Mitcheva M, & Belcheva A. (2015). Effects of Aronia melanocarpa Fruit Juice on Isolated Rat Hepatocytes. Pharmacognosy Magazine, 11(Suppl 4), S592-S597.

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Spleen Cell Isolation and Characterization

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Spleens were digested with collagenase from Clostridium histolyticum type IV (Sigma), deoxyribonuclease I from bovine pancreas grade II (Roche, IN, USA), and trypsin inhibitor type II-S soybean. Cells were washed with phosphate-buffered saline (Thermo Fisher Scientific) and stained with anti-CD11c (clone N418; BioLegend, CA, USA), anti-CD11b (clone M1/70; BioLegend), anti-CD8a (clone 53–6.7; BioLegend), anti-Ly6G (clone RB6–8C5; BioLegend), and anti-Ly6C (clone HK1.4; BioLegend). For measuring intracellular proteins, anti-phospho-NF-κB p65 (Ser⁵³⁶; clone 93H1; Cell Signaling, MA, USA) was used. For ROS measurement, cells were incubated with dihydrorhodamine 123 (100 μM; AnaSpec, CA, USA) for 30 min at 37°C.

Shaabani N., Honke N., Nguyen N., Huang Z., Arimoto K.I., Lazar D., Loe T.K., Lang K.S., Prinz M., Knobeloch K.P., Zhang D.E, & Teijaro J.R. (2018). The probacterial effect of type I interferon signaling requires its own negative regulator USP18. Science immunology, 3(27), eaau2125.

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Isolation and Characterization of Macrophages

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Silver nitrate (AgNO₃, 99%), l-glutathione (GSH, 98%), calcium chloride dihydrate, sodium hydroxide solution (NaOH, 1 N), collagenase from Clostridium histolyticum (type IV), and deuterium oxide (D₂O, 99.9%) were purchased from Sigma-Aldrich (St. Louis, MO). Anhydrous sodium sulfide (Na₂S) was purchased from Alfa Aesar (Tewksbury, MA). Hank’s Balanced Salt Solution (HBSS) buffer (1×) without calcium, magnesium, and phenol red was purchased from Invitrogen (Waltham, MA). EGTA buffer (0.5 M, pH 8) was purchased from Fisher Scientific (Pittsburgh, PA). OptiPrep density gradient medium (60% iodixanol in water) was purchased from BioVision Inc. (Milpitas, CA). An Insyte Autoguard shielded IV catheter (24G × 0.75”) and ethylene glycol were purchased from Becton, Dickinson and Company (Franklin Lakes, NJ). PE-conjugated monoclonal anti-F4/80 antibodies were purchased from Miltenyi Biotec (Bergisch Gladbach, North Rhine-Westphalia, Germany). Herringbone microfluidic chip mixers and male luer fluid connectors were purchased from Microfluidic ChipShop (Jena, Germany).

Hsu J.C., Du Y., Sengupta A., Dong Y.C., Mossburg K.J., Bouché M., Maidment A.D., Weljie A.M, & Cormode D.P. (2021). Effect of Nanoparticle Synthetic Conditions on Ligand Coating Integrity and Subsequent Nano-Biointeractions. ACS applied materials & interfaces, 13(49), 58401-58410.

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Isolation of Mouse Decidual and Spleen Cells

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Isolated mouse deciduas were minced with scissors and incubated for 30 min with 10 ml (1 mg/ml) of collagenase (collagenase from Clostridium histolyticum, type IV, Sigma-Aldrich, USA). The fragments were then passed through a 70-μm mesh and washed with RPMI1640 (Gibco, Life technologies, Scotland).
The pellet was resuspended in 10 ml of fetal calf serum (FCS)-free RPMI and filtered on a 40 μm filter. The cell count was adjusted to 1 × 10⁶/ml in RPMI1640 (Gibco, Life technologies, Scotland) +10% FCS (Gibco, Life Technologies, Scotland) + 1% penicillin/streptomycin (Gibco, Life Technologies, Scotland).
Spleen cells were isolated by passing the spleen through a 100-μm stainless steel mesh and centrifuging for 10 min at 1,000 rpm. The pellet was resuspended in 10 ml RPMI1640, filtered on a 70-μm mesh and further on a 40-μm mesh, washed, and resuspended in RPMI1640. The lymphocytes were isolated on Ficoll-Paque gradient, washed, and resuspended in RPMI1640 + 10% FCS + 1% penicillin/streptomycin.

Csabai T., Pallinger E., Kovacs A.F., Miko E., Bognar Z, & Szekeres-Bartho J. (2020). Altered Immune Response and Implantation Failure in Progesterone-Induced Blocking Factor-Deficient Mice. Frontiers in Immunology, 11, 349.

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Biochemical Analysis of Tissue Samples

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The following chemicals and reagents, including pentobarbital sodium (Sanofi, France), HEPES (Sigma Aldrich, Germany), NaCl (Merck, Germany), KCl (Merck), D-glucose (Merck), NaHCO3 (Merck), KH2PO4 (ScharlauChemie SA, Spain), K2HPO4 (ScharlauChemie SA, Spain), glycerol (Scharlau-Chemie SA, Spain), CaCl2x2H2O (Merck), MgSO4x 7H2O (Fluka AG, Germany), collagenase from Clostridium histolyticum type IV (Sigma Aldrich), albumin, bovine serum fraction V, minimum 98% (Sigma Aldrich), EGTA (Sigma Aldrich), 2-thiobarbituric acid (4,6-dihydroxypyrimidine-2-thiol; TBA) (Sigma Aldrich), trichloroacetic acid (TCA) (Valerus, Bulgaria), 2,2'-dinitro-5,5'-dithiodibenzoicacid (DTNB) (Merck), lactate dehydrogenase (LDH) kit (Randox, UK) were used.

Kondeva-Burdina M. (2020). QUANTITATIVE STRUCTURE-HEPATOTOXICITY ASSESSMENT OF SERIES ARYLPIPERAZINE-N1-SUBSTITUTED THEOBROMINE DERIVATIVES. FARMACIA, 68(1), 56-64.

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