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Collagenase x

Manufactured by Fujifilm
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Sourced in Japan

Collagenase X is a laboratory enzyme product manufactured by Fujifilm. It is a mixture of enzymes used for the digestion of collagen, a key structural component in various tissues. The core function of Collagenase X is to facilitate the breakdown and dissociation of collagen-rich samples, enabling downstream applications such as cell isolation and tissue dissociation.

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Lab products found in correlation

Hbss buffer, by Fujifilm (3 mentions) Medium 199, by Thermo Fisher Scientific (3 mentions) Hbss buffer, by Thermo Fisher Scientific (3 mentions) Trypsin edta, by Merck Group (1 mentions) Cell culture flask, by Corning (1 mentions) Fetal bovine serum (fbs), by Merck Group (1 mentions) Dispase 2, by Merck Group (1 mentions) Alpha modified eagle s medium, by Merck Group (1 mentions) C57bl 6j mice, by Sankyo Labo Service (1 mentions) Percoll solution, by GE Healthcare (1 mentions) Gw0742, by Bio-Techne (1 mentions) Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions) Wy14643, by Merck Group (1 mentions) William s e medium, by Thermo Fisher Scientific (1 mentions) Trizol, by Thermo Fisher Scientific (1 mentions) Rosiglitazone, by Bio-Techne (1 mentions)

Spelling variants of this product

Collagenase (247 citations) Collagenase type X (2 citations)

6 protocols using collagenase x

1

Isolation and Culture of Primary Mouse Osteoblasts

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The experimental procedures were approved by the Animal Committee of Tokyo Medical and Dental University. Primary osteoblast-like cells were collected from the cranial bones of neonatal littermates of C57BL/6 J mice (Sankyo Labo Service Co. Tokyo, Japan) and cultured as previously described [12 (link)]. In brief, the cranial bone was carefully cut up and digested with 0.1% collagenase X (Wako Pure Chemical Industries Ltd., Osaka, Japan), 0.1% dispase II (Sigma-Aldrich, St. Louis, MO, USA), and 0.05% trypsin–EDTA (Sigma-Aldrich, St. Louis, MO, USA) for 10 min at 37 °C. The suspension was then centrifuged to remove the supernatant, and the precipitate was resuspended and seeded in cell culture flasks (Corning, NY, USA) with alpha-Modified Eagle’s Medium (Sigma-Aldrich, MO, USA) medium containing 10% fetal bovine serum (Sigma-Aldrich, MO, USA) in an incubator with 5% CO2 at 37 °C.
Zhang M., Fukushima Y., Nozaki K., Nakanishi H., Deng J., Wakabayashi N, & Itaka K. (2023). Enhancement of bone regeneration by coadministration of angiogenic and osteogenic factors using messenger RNA. Inflammation and Regeneration, 43, 32.
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2

Isolation of Primary Hepatocytes

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For the isolation of primary hepatocytes, 8–10 weeks old wt and ob/ob mice were anesthetized by 2mg/ml xylazine and 2mg/ml ketamine. The liver was perfused through the portal vein with 10–15mL of warm (37°C) HBSS buffer (Invitrogen, CA) supplemented with 1 mM EGTA and 5mM of glucose, and then digested with 10–15mL of Collagenase X (WAKO, Japan) at 0.3mg/mL dissolved in HBSS buffer supplemented with 1.2mM CaCl2 and 5 mM glucose. After perfusion, primary hepatocytes were released and sedimented at 500 rpm for 2 minutes and washed two times with medium 199 (Invitrogen, CA). Hepatocytes were then layered on a 70% (for wt) and 50% (for ob/ob) Percoll gradient and centrifuged 2000 rpm for 10 minutes. The healthy cells were recovered at the bottom of the tube.
Arruda A.P., Pers B.M., Parlakgül G., Güney E., Inouye K, & Hotamisligil G.S. (2014). Chronic enrichment of hepatic ER-mitochondria contact sites leads to calcium dependent mitochondrial dysfunction in obesity. Nature medicine, 20(12), 1427-1435.
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3

Isolation of Murine Hepatocytes by Two-Step Perfusion

Cited 1 time
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Hepatocytes were isolated from liver of 12–14 weeks old L-Ago2 WT and L-Ago2 KO mice by a two-step perfusion method55 (link) with a slight modification. Briefly, the liver was first perfused with 30 ml of HBSS supplemented with 10 mM HEPES, 0.5 mM EGTA and 5 mM glucose and then digested with 35 ml of Collagenase X (WAKO) at 100 U ml−1 dissolved in HBSS buffer supplemented with 10 mM HEPES and 5 mM CaCl2. Liver was collected after perfusion and hepatocyte were released and sedimented at 60 × g for 2 min. Hepatocyte suspension was then layered on a 40% percoll solution (GE Healthcare Life Sciences) and centrifuged at 800 × g for 10 min. The alive hepatocytes were recovered from the bottom of the tube and seeded on culture plates.
Zhang C., Seo J., Murakami K., Salem E.S., Bernhard E., Borra V.J., Choi K., Yuan C.L., Chan C.C., Chen X., Huang T., Weirauch M.T., Divanovic S., Qi N.R., Thomas H.E., Mercer C.A., Siomi H, & Nakamura T. (2018). Hepatic Ago2-mediated RNA silencing controls energy metabolism linked to AMPK activation and obesity-associated pathophysiology. Nature Communications, 9, 3658.
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4

Isolation and Treatment of Primary Murine Hepatocytes

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Primary hepatocytes were isolated from L-Ago2 WT and KO mice using the perfusion method reported previously (34 ). Briefly, the mouse was anesthetized by isoflurane, and then an incision was made to expose the abdominal organs. A catheter (size 24-gauge) was inserted in the inferior vein cava to perfuse the liver with warm Hank’s balanced salt solution (HBSS) and again with warm HBSS containing collagenase X (Wako). The liver capsule was removed from the perfused liver, and the parenchymal cells were dispersed carefully into cold, low-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) in a cell culture dish. Then, the primary hepatocytes were purified using 40% density gradient buffer. After purification, the cells were cultured in William’s medium E (Invitrogen) for 2 to 3 hours and changed to 10% fetal bovine serum in low-glucose DMEM overnight. After the cells adhered to the plate, they were treated with 10-µM WY14643 (PPARα agonist, Sigma), 100-µM rosiglitazone (PPARγ agonist, Tocris), and 100-µM GW0742 (PPARδ agonist, Tocris) for 24 hours. Then, the cells were collected in Trizol (Invitrogen) for total RNA isolation.
Bhattacharjee J., Borra V.J., Salem E.S., Zhang C., Murakami K., Gill R.K., Kim A., Kim J.K., Salazar-Gonzalez R.M., Warren M., Kohli R, & Nakamura T. (2021). Hepatic Ago2 Regulates PPARα for Oxidative Metabolism Linked to Glycemic Control in Obesity and Post Bariatric Surgery. Endocrinology, 162(4), bqab007.
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5

Isolation of Primary Hepatocytes

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For the isolation of primary hepatocytes, 8–10 weeks old wt and ob/ob mice were anesthetized by 2mg/ml xylazine and 2mg/ml ketamine. The liver was perfused through the portal vein with 10–15mL of warm (37°C) HBSS buffer (Invitrogen, CA) supplemented with 1 mM EGTA and 5mM of glucose, and then digested with 10–15mL of Collagenase X (WAKO, Japan) at 0.3mg/mL dissolved in HBSS buffer supplemented with 1.2mM CaCl2 and 5 mM glucose. After perfusion, primary hepatocytes were released and sedimented at 500 rpm for 2 minutes and washed two times with medium 199 (Invitrogen, CA). Hepatocytes were then layered on a 70% (for wt) and 50% (for ob/ob) Percoll gradient and centrifuged 2000 rpm for 10 minutes. The healthy cells were recovered at the bottom of the tube.
Arruda A.P., Pers B.M., Parlakgül G., Güney E., Inouye K, & Hotamisligil G.S. (2014). Chronic enrichment of hepatic ER-mitochondria contact sites leads to calcium dependent mitochondrial dysfunction in obesity. Nature medicine, 20(12), 1427-1435.
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6

Isolation of Murine Primary Hepatocytes

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Male mice were anesthetized using 2mg/ml xylazine combined with 2mg/ml ketamine in PBS and the liver perfused through the portal vein with 10–15mL warm (37°C) HBSS buffer (Invitrogen, CA) supplemented with 10mM Hepes, 1 mM EGTA and 5mM of glucose, pH 7.4. Next, the liver were perfused with 20mL of HBSS buffer supplemented with 10mM Hepes, 1.2mM CaCl2 and 5 mM glucose, in the presence of 0.3mg/mL Collagenase X (WAKO, Japan) in tissue digestion. The buffers were perfused at a speed of 3mL/min. After digestion, the hepatocytes were carefully released and sedimented at 500 rpm for 2 minutes and washed two times with medium 199 (Invitrogen, CA). The hepatocytes were then homogenized in Williams E medium in the presence of 5% CCS. To separate live from dead cells, the hepatocyte suspension was layered on a 30% Percoll gradient and centrifuged 1500 rpm for 10 minutes. The healthy cells were recovered at the bottom of the tube and plated in Williams E medium supplemented by 5% CCS and 1mM glutamine for experimentation. All experiments in primary hepatocytes were conducted within 24 hours of isolation and plating in order to maintain the Cx expression pattern as closed as possible to the in vivo setting.
Tirosh A., Tuncman G., Calay E.S., Rathaus M., Ron I., Tirosh A., Yalcin A., Lee Y.G., Livne R., Ron S., Minsky N., Arruda A.P, & Hotamisligil G.S. (2020). Inter-cellular transmission of hepatic ER stress in obesity disrupts systemic metabolism. Cell metabolism, 33(2), 319-333.e6.
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