Collagenase l

Manufactured by Nitta Gelatin

Sourced in Japan

Collagenase L is a laboratory enzyme used to break down collagen, a structural protein found in various tissues. It is primarily used in research and experimental settings to facilitate the isolation and study of cells and tissues.

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Lab products found in correlation

Midimacs column, by Miltenyi Biotec (1 mentions) Cd11b microbead, by Miltenyi Biotec (1 mentions) Facscalibur flow cytometer, by BD (1 mentions) Dnase 1, by Roche (1 mentions) Cellquest software program, by BD (1 mentions) Hyaluronidase h3506, by Merck Group (1 mentions) Protease inhibitor cocktail, by Nacalai Tesque (1 mentions) 75 cm2 flask, by BD (1 mentions) Trypsin, by Merck Group (1 mentions) Gentamicin sulfate, by Fujifilm (1 mentions) Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions) Monothioglycerol, by Fujifilm (1 mentions) L alanyl l glutamine, by Fujifilm (1 mentions) Rpmi 1640, by Thermo Fisher Scientific (1 mentions) Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions) Fv500 confocal microscope, by Olympus (1 mentions) Dispase, by Thermo Fisher Scientific (1 mentions) Lipofectamine ltx with plus reagent, by Thermo Fisher Scientific (1 mentions) Fluoview software, by Olympus (1 mentions) Smartspec plus, by Bio-Rad (1 mentions)

7 protocols using collagenase l

Isolation and Characterization of Tumor-Associated Macrophages

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CD11b+ cells were isolated from mouse xenograft tumors or Matrigel plugs by magnetic sorting using CD11b MicroBeads (Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany). In brief, tissues minced in PBS were incubated in collagenase L (Nitta Gelatin, Osaka, Japan) and DNase I (Roche Diagnostics GmbH, Mannheim, Germany) at final concentrations of 0.5% and 20 unit/mL. The mixture was incubated for 1 h at 37°C under gentle agitation. Digestion was stopped using fetal bovine serum, after which the cell suspension was washed and then passed through a 70-µm-mesh nylon screen. The cells were incubated with CD11b MicroBeads for 15 min at 4°C and loaded onto a MIDIMACS column (Miltenyi Biotec GmbH) according to the manufacturer's instructions. Isolated CD11b+ cells from xenografts or Matrigel were used as TAMs for further experiments. For cell surface staining, single-cell suspensions were incubated with fluorescein isothiocyanate–conjugated anti-CD11b monoclonal antibody (BD Bioscience) and phycoerythrin-conjugated anti-F4/80 monoclonal antibody (eBioscience, Inc., San Diego, CA) for 15 min at 4°C. The stained cells were run on a FACSCalibur flow cytometer (BD Biosciences). The data were analyzed using the CellQuest software program (BD Biosciences).

Watari K., Shibata T., Kawahara A., Sata K.I., Nabeshima H., Shinoda A., Abe H., Azuma K., Murakami Y., Izumi H., Takahashi T., Kage M., Kuwano M, & Ono M. (2014). Tumor-Derived Interleukin-1 Promotes Lymphangiogenesis and Lymph Node Metastasis through M2-Type Macrophages. PLoS ONE, 9(6), e99568.

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Glucagon Secretion in Pancreatic Islets

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Pancreatic islets were isolated from Hnf1a+/+ and Hnf1a−/− mice as previously described [16 (link)]. Briefly, after bile duct cannulation and digestion of the pancreas using a mixture of 1.5 mg/mL collagenase L (Nitta Gelatin, Osaka, Japan), 1.5 mg/mL hyaluronidase (H3506; Sigma-Aldrich, St. Louis, MO), and 0.1% (v/v) protease inhibitor cocktail (Nacalai Tesque, Inc., Kyoto, Japan), isolated islets were manually collected. Islet glucagon was extracted by an acid–ethanol extraction method as previously described [17 (link)]. To assess glucagon secretion from isolated islets in vitro, size-matched islets were pre-incubated in pH 7.4 HEPES-buffered Krebs-Ringer bicarbonate solution (KRBH) buffer (120 mM NaCl, 4.7 mM KCl, 1.2 mM KH₂PO₄, 2.4 mM CaCl₂, 1.2 mM MgCl₂, 20 mM NaHCO₃, and 10 mM HEPES) containing 11 mM glucose and then stimulated with KRBH buffer containing 1.1 or 5.6 mM glucose, or with 20 mM KCl for the indicated time. To examine the effect of SGLT inhibition on glucagon secretion, isolated islets were pre-incubated with 20 μM sotagliflozin for 2 h, after which a glucagon secretion assay was performed in the presence of 20 μM sotagliflozin. Glucagon concentration was determined by a mouse glucagon enzyme-linked immunosorbent assay (ELISA) kit (Mercodia AB, Uppsala, Sweden) and glucagon levels were normalized by islet number.

Sato Y., Rahman M.M., Haneda M., Tsuyama T., Mizumoto T., Yoshizawa T., Kitamura T., Gonzalez F.J., Yamamura K.I, & Yamagata K. (2020). HNF1α controls glucagon secretion in pancreatic α-cells through modulation of SGLT1. Biochimica et biophysica acta. Molecular basis of disease, 1866(11), 165898.

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Chondrocyte Isolation and Monolayer Culture

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Articular cartilage specimens, collected and minced into small pieces as above, were incubated with 0.015% trypsin (Sigma-Aldrich, St. Louis, MO, USA) for 15 min, and subsequently digested with 0.25% collagenase (Collagenase L; Nitta Gelatin, Osaka, Japan) for 4 h at 37 °C [34 (link)]. The isolated chondrocytes were cultured as monolayers in 75 cm² flasks (Falcon, Lincoln, NY, USA) in complete DMEM for one week at 37 °C in 5% CO₂/95% humidified air.

Ichimaru S., Nakagawa S., Arai Y., Kishida T., Shin-Ya M., Honjo K., Tsuchida S., Inoue H., Fujiwara H., Shimomura S., Mazda O, & Kubo T. (2016). Hypoxia Potentiates Anabolic Effects of Exogenous Hyaluronic Acid in Rat Articular Cartilage. International Journal of Molecular Sciences, 17(7), 1013.

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Culturing INS1E and Rat Pancreatic Fibroblasts

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The INS1E cell line, a gift from Prof. Pierre Maechler,¹⁴ (link) was maintained in RPMI-1640 (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal calf serum (Gibco), 0.5 mM monothioglycerol (Antioxidant, Wako, Osaka, Japan), 2 mM L-alanyl-L-glutamine (Wako), and 50 μg/ml gentamicin sulfate (Wako). Rat pancreatic fibroblasts were cultured under the same conditions as INS1E cells. Rat neonatal pancreases were collected and enzymatically digested in a collagenase/proteinase cocktail (0.1% collagenase L, Nitta Gelatin, Osaka, Japan, 0.2% dispase II, Gibco in HBSS, Gibco) for 60 mins in a reciprocating water bath shaker at 37°C. Undigested debris was removed using a nylon mesh and the cells were cultured as described below. Attached fibroblasts proliferated continuously and were sequentially passaged (sub-cultured in another dish), effectively diluting any contaminating islets and blood cells. Pancreatic fibroblasts exhibited cellular senescence after 3 to 6 passages, and were used before cell division had slowed down.

Tago K., Inoue K.I., Ouchi M., Miura Y, & Kubota K. (2016). Receptor for advanced glycation endproducts signaling cascades are activated in pancreatic fibroblasts, but not in the INS1E insulinoma cell line: Are mesenchymal cells major players in chronic inflammation?. Islets, 8(5), 135-144.

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Isolation and Transfection of Mouse Ear Fibroblasts

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The ear tips derived from 2-week-old mice were diced into small pieces, incubated at 37°C for 30 minutes with 4 mg/ml collagenase L (Nitta Gelatin, Naniwa, Osaka, Japan) and 4 mg/ml dispase (Life Technologies), and then cultured with 10% fetal bovine serum/Dulbecco’s modified eagle medium at 37°C and 10% CO₂ for several days. pCAG-Cre, pCMV-tTA (Takara), and pCMV-DsRed (Takara) were co-transfected into primary fibroblast cells in a six-well plate using Lipofectamine LTX with Plus reagent (Life Technologies). Images were acquired on a FV500 confocal microscope and Fluoview software (Olympus, Shinjuku, Tokyo, Japan).

Aida T., Chiyo K., Usami T., Ishikubo H., Imahashi R., Wada Y., Tanaka K.F., Sakuma T., Yamamoto T, & Tanaka K. (2015). Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice. Genome Biology, 16(1), 87.

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3D Cell Culture Proliferation Assay

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Cells (5 × 10⁴) were seeded on a culture dish (radius of 17.5 mm) filled with 500 μL of collagen gel. One day after seeding, 250 μL of collagen sol solution was poured on the cells and incubated for approximately 30 min at 37°C to induce gelation. The dish was then filled with culture medium and incubated at 37°C. After 1 day, cells were randomly imaged under a phase-contrast microscope (TE300; Nikon Instech Co.) with a 10× objective, and the colony size was determined by calculating the area of the colonies using ImageJ software (National Institutes of Health, Bethesda, MD). After 1, 2, or 3 day(s), the cells were treated with 0.1% collagenase-L (Nitta Gelatin) in PBS and incubated for 1 h at 37°C. After incubation, the cells were collected and suspended in 500 μL of trypsin-EDTA. The OD600 was calculated using an absorption spectrometer (SmartSpec Plus; Bio-Rad), and the cell number was determined according to the following formula: cell number = OD600 × 6.85 × 10⁵. Alternatively, cell number was directly counted by a counting chamber.

Ishihara S., Yasuda M., Ishizu A., Ishikawa M., Shirato H, & Haga H. (2015). Activating transcription factor 5 enhances radioresistance and malignancy in cancer cells. Oncotarget, 6(7), 4602-4614.

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Adult Mouse Salivary Gland Analysis

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Male C57BL/6J adult mice (8 weeks of age) were purchased from CLEA Japan (Tokyo, Japan). Mice were maintained under a 12-h light/dark cycle and fed ad libitum. All experiments were approved by the Animal Committee of Kyushu Dental University (No. 16–011). Collagenase L was purchased from Nitta Gelatin (Osaka, Japan); Fura 2-AM was purchased from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan); 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) was purchased from Life Technologies Corporation (Eugene, OR, USA); T16Ainh-A01 was obtained from Tocris Bioscience (Bristol, UK); the NKCC1 antibody (sc-21545) was obtained from Santa Cruz Biotechnology (Dallas, TX, USA); the TMEM16A antibody (ab53213) was purchased from Abcam (Cambridge, UK); the AQP5 antibody (sc-9890) was purchased from EMD Millipore (Billerica, MA, USA); and the secondary antibody (Histofine Simple Stain Mouse MAX peroxidase) was purchased from Nichirei (Tokyo, Japan). All other reagents were purchased from Sigma-Aldrich Japan (Tokyo, Japan).

Kusuda Y., Kondo Y., Miyagi Y., Munemasa T., Hori Y., Aonuma F., Tsuka S., Mukaibo T., Masaki C, & Hosokawa R. (2019). Long-term dexamethasone treatment diminishes store-operated Ca2+ entry in salivary acinar cells. International Journal of Oral Science, 11(1), 1.

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