The Ethics Committee of the Medical Research Council of Hungary (ETT-TUKEB, No. 51323-4/2015/EKU) approved all experiments with human samples and informed consent was obtained from the patients. Samples were collected at the Department of Oncosurgery, Uzsoki Hospital, Budapest, Hungary. Patient data are shown in Supplementary Tables S1 , S2 . Tumor and normal colon tissue dissected at a distance >5 cm from the tumor were isolated and cut into small pieces (<0.5 cm) in PBS. After extensive washing with PBS three times, tissue pieces were incubated in a digestive mix [DMEM high glucose with 20% FBS, 75 U/mL collagenase type IX (Sigma), and 125 μg/mL dispase type II (Invitrogen, Carlsbad, CA, United States)] for 2 h at 37°C with extensive shaking. After removal of tissue pieces, single cells were then centrifuged at 300 g for 5 min, washed twice in PBS and cultured in tissue culture plates (Eppendorf) in fibroblast medium containing 15% FBS.
Collagenase type 9
Manufactured by Merck Group
Sourced in United States, Italy
Collagenase type IX is a digestive enzyme used in laboratory research. It is derived from Clostridium histolyticum and has the ability to break down collagen, a structural protein found in various tissues. This enzyme is commonly used in cell isolation and tissue dissociation procedures.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Dispase type 2, by Thermo Fisher Scientific (2 mentions)
Mem vitamin solution, by Merck Group (2 mentions)
Earle s balanced salt solution, by Merck Group (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Tissue culture plates, by Eppendorf (1 mentions)
Matrigel, by Corning (1 mentions)
40 μm cell strainer, by BD (1 mentions)
12 mm diameter coverslips, by Matsunami (1 mentions)
Fetal bovine serum (fbs), by Euroclone (1 mentions)
L glutamine, by Euroclone (1 mentions)
5 protocols using collagenase type 9
1
Isolation and culture of colon cancer cells
2
Isolation of Metastatic CRC Tumor Cells
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Tumor samples from metastatic CRC patients were collected under a Duke IRB approved protocol (Pro00002435) at Duke University Hospital. All participants provided written informed consent to participate in the study. Tumor samples were chopped into 5 mm pieces and washed with PBS several times. The tumor fragments were incubated in digestion buffer (Dulbecco's modified Eagle medium with 2.5% fetal bovine serum, penicillin/streptomycin [Invitrogen], 75 U/mL collagenase type IX [Sigma], 125 μg/mL dispase type II [Invitrogen]) for 60 min at 37 °C. The supernatant was collected in a 50 mL Falcon tube, centrifuged at 1000RPM for 5 min, and then washed with PBS repeatedly. Isolated cancer cells were counted using a hemocytometer. Single cells were embedded in ice cold Matrigel (Corning Life Sciences) and seeded in 24-well plates. Matrigel was polymerized for 10 min at 37 °C. Basal culture medium was supplemented with a combination of growth factors as previously described.13 (link)
3
Isolation of Mouse Dorsal Root Ganglion Neurons
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After anesthesia with isoflurane, the dorsal root ganglion (DRG) was separated from the L4 to L6 of mice after perfusion with 10 mL ice-cold artificial cerebrospinal fluid (aCSF: 124 mM NaCl, 5 mM KCl, 1.2 mM KH2PO4, 1.3 mM MgSO4, 2.4 mM CaCl2, 10 mM glucose, 24 mM NaHCO3, and equilibrated with 95% O2 and 5% CO2 for 1 hour on ice). The tissues were incubated with 725 µg of collagenase type IX (lot# SLBG3258; Sigma-Aldrich) in 250 µL of Earle's balanced salt solution (Sigma-Aldrich) containing 10% FBS (as above), MEM vitamin solution (1:100, Sigma-Aldrich), penicillin-streptomycin (1:200, Life Technologies, Carlsbad, CA), and GlutaMax (1:100, Life Technologies) at 37°C for 25 minutes. Next, the DRG neurons were mechanically separated by 10 to 20 cycles of pipetting using a small diameter Pasteur pipette and filtered through a 40-μm cell strainer (BD Falcon, Franklin Lakes, NJ). The isolated neurons were placed on 12-mm diameter coverslips (Matsunami, Japan) with 40 µL of aCSF and used for experiments within 4 hours after isolation, maintaining them at 37°C in a 95% O2 and 5% CO2 humidified chamber.
4
Isolation of Murine Bone and Muscle Cells
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The bone and skeletal muscle tissues were collected from wild-type male and CS mice described above [9 (link),12 (link)]. Briefly, the muscles were incubated with Dulbecco’s modified Eagle’s medium (DMEM+) solution (EuroClone S.p.A., Milano, Italy) composed of 1X antibiotics (1%), L-glutamine (1%), FBS (10%) (EuroClone, S.p.A., Milano, Italy) and enriched with collagenase type IX (0.1%) (SIGMA Chemical Co., Milan, Italy). The fibers were then enzymatically isolated from the Flexor digitorum brevis (FDB) mice muscles in the shaker incubator for 2 h [9 (link),30 (link),31 (link)].
The culture of primary bone cells from adult mice was obtained from long bone such as femora or calvaria, as previously described [9 (link),31 (link)] Bones were collected, cleaned, and flushed to remove the internal bone marrow cells. Small bone pieces of around 1 mm3 were treated with trypsin-EDTA 0.25% (w/v) for 1 h and with 0.2% collagenase solution for an additional hour in a shaking water bath to remove all remaining soft tissue and adherent cells. Clean bone pieces have been cultured in a basal medium enriched with 50 µg/mL of ascorbic acid. As previously described, osteoblasts from calvaria chips were positive for Alizarin red staining and positive for PCR gene expression on cell pellets [32 (link)].
The culture of primary bone cells from adult mice was obtained from long bone such as femora or calvaria, as previously described [9 (link),31 (link)] Bones were collected, cleaned, and flushed to remove the internal bone marrow cells. Small bone pieces of around 1 mm3 were treated with trypsin-EDTA 0.25% (w/v) for 1 h and with 0.2% collagenase solution for an additional hour in a shaking water bath to remove all remaining soft tissue and adherent cells. Clean bone pieces have been cultured in a basal medium enriched with 50 µg/mL of ascorbic acid. As previously described, osteoblasts from calvaria chips were positive for Alizarin red staining and positive for PCR gene expression on cell pellets [32 (link)].
5
Isolation of Dorsal Root Ganglion Neurons from Mice
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According to a published method49 with some minor modifications described as follows, DRG were isolated from 5 to 10 week old mice. In brief, resected DRG were collected in PBS (calcium- and magnesium-free) on ice, and then the tissues were incubated with 725 μg of collagenase type IX (catalog No. C9407, Sigma-Aldrich) in 250 μL of Earle’s balanced salt solution (Sigma-Aldrich) containing 10% fetal bovine serum, MEM vitamin solution (1:100, Sigma-Aldrich), penicillin–streptomycin (1:200, Life Technologies), and GlutaMax (1:100, Life Technologies) at 37 °C for 30 min. Next, the DRG neurons were dissociated by triturating the suspension through a fire-polished Pasteur pipette and filtering it through a 70 μm cell strainer (Flowmi). The isolated neurons were placed on 12 mm diameter coverslips (Matsunami, Osaka, Japan) with 20 μL of Earle’s balanced salt solution and used for experiments within 2 h of isolation, maintaining them at 37 °C in a chamber under a humidified atmosphere of 95% O2 and 5% CO2.
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