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Collagenase

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Collagenase is an enzyme that breaks down collagen, a structural protein found in connective tissues. It is commonly used in laboratory applications to isolate and harvest cells from tissues.

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Lab products found in correlation

Trypsin, by Thermo Fisher Scientific (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions) Sodium azide nan3, by Merck Group (1 mentions) Summit 4, by Beckman Coulter (1 mentions) Collagenase, by Merck Group (1 mentions) Pronase, by Merck Group (1 mentions) Dnase, by MP Biomedicals (1 mentions) Lysozyme, by MP Biomedicals (1 mentions)

6 protocols using collagenase

1

Isolation and Cloning of MEFs and Tumor Cells

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To generate clonal MEFs, we isolated E13.5-E14.5 aged embryos, removed fetal heart and liver structures, and digested remaining tissue both mechanically and enzymatically with 0.1% Trypsin (GIBCO) for 5 minutes at 37°C. Cell suspension from digested tissue was cultured overnight in DMEM supplemented with 10% FBS. Adherent cells were serially diluted and plated in 96-well plates. Single cell clones were identified and expanded.
To generate the tumor-derived cell lines, a ~ 10mm3 tumor piece was isolated at necropsy. The tumor was mechanically and enzymatically digested with 2mg/mL Collagenase (MP Biomedical) in DMEM supplemented with 10% FBS for 30 minutes at 37°C. All tumor tissues were cultured overnight in DMEM supplemented with 10% FBS. Adherent cells were isolated, serially diluted, and plated in 96-well plates. Single cell clones were identified and expanded.
Nagdas S., Kashatus J.A., Nascimento A., Hussain S.S., Trainor R.E., Pollock S.R., Adair S.J., Michaels A.D., Sesaki H., Stelow E.B., Bauer T.W, & Kashatus D.F. (2019). Drp1 Promotes KRas-Driven Metabolic Changes to Drive Pancreatic Tumor Growth. Cell reports, 28(7), 1845-1859.e5.
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2

Isolation and Cloning of MEFs and Tumor Cells

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To generate clonal MEFs, we isolated E13.5-E14.5 aged embryos, removed fetal heart and liver structures, and digested remaining tissue both mechanically and enzymatically with 0.1% Trypsin (GIBCO) for 5 minutes at 37°C. Cell suspension from digested tissue was cultured overnight in DMEM supplemented with 10% FBS. Adherent cells were serially diluted and plated in 96-well plates. Single cell clones were identified and expanded.
To generate the tumor-derived cell lines, a ~ 10mm3 tumor piece was isolated at necropsy. The tumor was mechanically and enzymatically digested with 2mg/mL Collagenase (MP Biomedical) in DMEM supplemented with 10% FBS for 30 minutes at 37°C. All tumor tissues were cultured overnight in DMEM supplemented with 10% FBS. Adherent cells were isolated, serially diluted, and plated in 96-well plates. Single cell clones were identified and expanded.
Nagdas S., Kashatus J.A., Nascimento A., Hussain S.S., Trainor R.E., Pollock S.R., Adair S.J., Michaels A.D., Sesaki H., Stelow E.B., Bauer T.W, & Kashatus D.F. (2019). Drp1 Promotes KRas-Driven Metabolic Changes to Drive Pancreatic Tumor Growth. Cell reports, 28(7), 1845-1859.e5.
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3

Generation and Characterization of Murine Pancreatic Tumor Cell Lines

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The generation of murine pancreatic tumor cell lines KPDC253, KPDC145 and KPDC143 was described previously34 (link). Briefly, a ~ 10 mm3 tumor piece was isolated at necropsy from mice with genetically-induced pancreatic ductal adenocarcinoma. The tumor was mechanically and enzymatically digested with 2 mg/mL Collagenase (MP Biomedical) in DMEM supplemented with 10% FBS for 30 min at 37 °C. All tumor tissues were cultured overnight in DMEM supplemented with 10% FBS. Adherent cells were isolated, serially diluted, and plated in 96-well plates. Single cell clones were identified and expanded and the Drp1 genotype was confirmed by PCR. In accordance with institutional guidelines, all mice used in the generation of these cell lines were monitored and euthanized when they reached pre-determined endpoints or exhibited features associated with disease such as weight loss. All animal studies and procedures were approved by the University of Virginia Institutional Animal Care and Use Committee.
Rohani A., Kashatus J.A., Sessions D.T., Sharmin S, & Kashatus D.F. (2020). Mito Hacker: a set of tools to enable high-throughput analysis of mitochondrial network morphology. Scientific Reports, 10, 18941.
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4

Flow Cytometric Analysis of Fibroblast and Endothelial Cells

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Samples were collected at day 0, 7 and 14, washed twice in PBS and digested in 1 mL of 1.0 mg/mL collagenase (MP Biomedical, Santa Ana, CA) in Ca2+/Mg2+ free HBSS solution at 37°C in a humidified chamber for 60 min to obtain a single cell suspension. For flow analysis, GFP-transduced FB were used. MVEC were labelled with the endothelial cell-specific marker UEA-I conjugated to rhodamine (Vector Laboratories) and dead cells were excluded using 2 ng/mL DAPI (Molecular probes). All cell labelling was done in the dark on ice in cold FACS buffer (PBS, 7% FBS, 0.1% NaN3 sodium azide (sigma)). The samples were then washed twice in PBS, filtered through 80 µm nylon mesh and analyzed in a Coulter Cyan #5 analyzer (Beckman Coulter, Miami, FL). Data were exported as FCS files and analyzed using Summit 4.3 (Beckman Coulter, Inc. Fullerton, CA). Pure populations of GFP transduced FB and MVEC were used as positive control.
Tiruvannamalai Annamalai R., Rioja A.Y., Putnam A.J, & Stegemann J.P. (2016). Vascular Network Formation by Human Microvascular Endothelial Cells in Modular Fibrin Microtissues. ACS biomaterials science & engineering, 2(11), 1914-1925.
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5

Isolation and Activation of Hepatic Stellate Cells

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HSCs were isolated from male BALB/c mice (14 weeks old) as described previously41 (link) with some modifications. In brief, the livers were perfused in situ with phosphate-buffered saline (PBS) and then with Gey’s balanced salt solution (GBSS) supplemented with collagenase (0.5 mg/ml; Sigma-Aldrich) and pronase (1 mg/ml; Sigma-Aldrich). The perfused livers were dissected, and the attached gall bladders and connective tissues were removed. The liver cell suspensions were further digested in GBSS supplemented with collagenase (0.25 mg/ml), pronase (0.5 mg/ml), and DNase (0.07 mg/ml; MP Biomedicals, Santa Ana, CA, USA), for 12 min in a 37 °C water bath. The cells were then washed and centrifuged in a 13.4% Nycodenz gradient at 1400×g for 20 min without brake. The interface containing the enriched HSCs was collected and washed with GBSS. Then, the isolated HSCs were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. The purity of the HSCs was assessed by microscopic observation. The HSCs were passaged before reaching 70% confluence in the primary culture and used as activated HSCs. The activation status of the HSCs was assessed on the basis of their increased expression of α-SMA and collagen type I as well as through their morphologic changes.
Park J.H., Kim J., Choi S.Y., Lee B., Lee J.E., Park H., Moon J.W., Park S.H., Lee J.M., Lee H.S, & Oh J. (2021). Albumin inhibits the nuclear translocation of Smad3 via interleukin-1beta signaling in hepatic stellate cells. Scientific Reports, 11, 3196.
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6

Enzymatic Degradation of Polymer Gels

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All LIMB samples were prepared of the same size and weighed at Day 0. After that, an enzyme solution consisting of 50 µg/mL collagenase (MP Biomedicals, 1951091), 50 µg/mL lysozyme (MP Biomedicals, 100831), and 0.01% sodium azide (NaN3, Sigma, S2002) in PBS was added to the gels. The samples were incubated at 37 °C with gentle mechanical stimulation at 75 rpm over 35 days. The enzyme solution was changed every other day. At pre-determined time intervals, the enzyme solution was removed. The samples were then washed three times (5 min each wash) with deionized water. The samples were then lyophilized, and the remaining polymer dry weight was measured. The remaining ratio of the polymer was calculated by dividing the remaining polymer dry weight by the initial dry weight of gels.
Bao G., Gao Q., Cau M., Ali-Mohamad N., Strong M., Jiang S., Yang Z., Valiei A., Ma Z., Amabili M., Gao Z.H., Mongeau L., Kastrup C, & Li J. (2022). Liquid-infused microstructured bioadhesives halt non-compressible hemorrhage. Nature Communications, 13, 5035.
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