Fibroblasts were seeded at a density of 2.5 million cells/ml as previously described (31 (link)). Fibroblasts were seeded in DMEM with 10% FCS and 1% PS at a density of 2.5 million cells/ml on the corners of a 1 cm2 piece of Matriderm (MedSkin Solutions), meaning 250 000 cells per piece of Matriderm. Two days after seeding, the medium was changed to DMEM supplemented with 2% heat-inactivated FCS, 1% PS and 5 ng/ml human recombinant TGFβ1 (4342-5, BioVision). Heat inactivation was performed by placing the FCS in a 56°C water bath for 30 min and mixing regularly. Medium supplemented with TGFβ1 was changed every 4 days. After 14 days, the cells were enzymatically extracted from the Matriderm with the use of a collagenase solution in complete medium (2000 IU/ml, Worthington) at 37°C on a shaker for 3 h. Centrifugation was performed to remove collagenase and cells were transferred to new flasks and cultured for further experiments. Passage number of ‘VSMC-like’ cells after transdifferentiation was kept below five to prevent loss of transdifferentiation markers.
Collagenase solution
Manufactured by Worthington
Sourced in United States
Collagenase solution is a laboratory product used to break down collagen, a structural protein found in various tissues. It is commonly used in cell biology and tissue engineering applications to facilitate the isolation and extraction of cells from their extracellular matrix.
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Lab products found in correlation
Dulbecco s modified eagle s medium d mem nutrient mixture f 12 ham, by Merck Group (1 mentions)
Stabilized antibiotic antimycotic solution 100, by Merck Group (1 mentions)
Trypsin edta 1, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Biowest (1 mentions)
Cell strainer, by Corning (1 mentions)
Primeflow rna assay kit, by Thermo Fisher Scientific (1 mentions)
Grace s medium, by Merck Group (1 mentions)
Gentamycin, by Sartorius (1 mentions)
F7524, by Merck Group (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
D2650, by Merck Group (1 mentions)
C57bl 6j mice, by Jackson ImmunoResearch (1 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (1 mentions)
Endothelial cell growth factor, by Merck Group (1 mentions)
Heparin, by Merck Group (1 mentions)
M199 media, by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
10 protocols using collagenase solution
1
Matriderm-based Fibroblast Transdifferentiation
2
Isolation and Expansion of Perichondrial Cells
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We obtained elastic cartilage samples from microtia patients following the approved guidelines set by the ethical committee at Yokohama City University Graduate School of Medicine Hospital (approval no. B130905006). We stripped off the adipose tissue and microscopically separated the cartilage into three layers: the chondrium layer, interlayer, and perichondrium layer. The perichondrial sample was minced with scissors until almost no lumps of the cartilage matrix were found and the perichondrial cells were separated by shaking in a 0.2% collagenase solution (Worthington, Lakewood, NJ, USA) at 37 °C and 600 rpm for 2 h. The resulting suspension was filtered through a 40 µm cell strainer and centrifuged at 4 °C and 1500 rpm for 5 min to collect perichondrocytes. Having been suspended in growth medium and seeded on an uncoated 35 mm dish, the final concentration contained 10% fetal bovine serum (Biowest, Riverside, MO, US), Dulbecco’s Modified Eagle’s Medium (D-MEM)/Nutrient Mixture F-12 Ham (1:1) (Sigma-Aldrich, St. Louis, MO, US), and stabilized antibiotic–antimycotic solution (100×) (Sigma-Aldrich, St. Louis, MO, US). When the perichondrocytes adhered and became confluent, the cells were collected using 0.05% trypsin–EDTA (1×) (Thermo Fisher Scientific, Waltham, MA, US) and expanded in a 225 cm2 flask (n = 4).
3
Isolation of Adult Mouse Ventricular Cardiomyocytes
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All procedures were in accordance with New York University guidelines for animal use and care (IACUC Protocol 130812-01 to MD approved on 08/13/2014) and conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication 58-23, revised 1996). Experiments were carried out in C57BI/6 adult mice of both genders (3–6 months of age). Adult mouse ventricular myocytes were obtained by enzymatic dissociation following standard procedures. Briefly, mice were injected with 0.1 ml heparin (500 IU ml−1 intraperitoneally) 20 min before heart excision and anaesthetized by carbon dioxide inhalation. Deep anaesthesia was confirmed by lack of response to otherwise painful stimuli. Hearts were quickly removed from the chest and placed in a Langendorff column. The isolated hearts were perfused sequentially with low calcium, and collagenase solution (Worthington). Atria were discarded and ventricles were gently minced with a Pasteur pipette. Calcium concentration was reintroduced gradually to normal values. After isolation, cardiomyocytes were plated on laminin coated coverslips or dishes and left to adhere for at least 30 min before the start of experiments.
4
Isolation and Culture of Porcine Valve Interstitial Cells
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Valve interstitial cells for in vitro culture were isolated from fresh porcine hearts obtained from a local abattoir (Cockrum’s Custom Meat Processing and Taxidermy, AR) using techniques published by us previously [14 (link), 19 (link), 20 (link)]. Briefly, the heart was transported to the laboratory in ice-cold, sterile PBS solution and quickly dissected in the laboratory using aseptic techniques. All three aortic valve leaflets were dissected and incubated in 1 mg/ml collagenase solution (Worthington, NJ) for 3 h at 37 °C with frequent agitation. After collagenase digestion, cold 10% fetal bovine serum (FBS)-containing Dulbecco’s Modified Eagle Media (DMEM) was added to arrest enzymatic activity. The solution was filtered with the cell strainer 100 μm pore size (Corning, NY) to remove any remaining tissue debris prior to centrifugation for 5 min at 200 g and 4 °C. The resulting cell pellet was re-suspended in 10% FBS-containing cell culture media, plated in a flask and maintained in a 37 °C incubator. Fresh media was changed at least every 3 days. Cells from passage 1–7 were used in all subsequent two-dimensional (2D) and three-dimensional (3D) culture studies.
5
Microgel Degradation Kinetics
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A stock solution was prepared by adding a precise amount of dehydrated and lyophilized microgels in 1X DPBS (stock = 0.67 mg/mL). 200 μL of the sock solution (~130 μg/well) was dispensed in a 96 well plate, and an equal volume of collagenase solution (Worthington Biochemical) was added to the wells at different concentrations and sealed with a clear plate cover to prevent evaporation. The fluorescence of the supernatant was then measured at Ex/Em 590/620 every 5 min for 48 hours to assess microgel degradation rates.
6
Isolation and Sorting of Drosophila Ovarian Cells
7
Biobanking Human Breast Tissue
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The use of human tissue has been approved by the Scientific Ethical Committee of Region Hovedstaden and the Danish Data Protection Agency with reference to H-2-2011-052 and 2011-41-6722, respectively, and patients agreed to donate tissue by written consent. Normal breast tissue was acquired from 29 female donors undergoing reduction mammoplasty for cosmetic purposes. Donors remain anonymous except their ages at the time of surgery. 36 breast carcinoma specimens were donated by women undergoing mastectomy for primary breast cancer. Tissue was cut into pieces for cryo-sectioning or cut finely prior to dissociation using 900 U/ml collagenase solution (Worthington Biochemical) in DMEM/F12 (Gibco) supplemented with 2 mM glutamine and 50 μg/ml gentamycin (Biological Industries) to release epithelial organoids, upon collagenase digestion comprised of epithelium and adjacent stromal cells82 , which were then stored in liquid nitrogen with 90% fetal bovine serum (F7524, Sigma-Aldrich) and 10% dimethyl sulfoxide (D2650, Sigma-Aldrich), which we find, is the optimal condition for freezing, thawing and survival83 (link). Some of the biopsies used in this study have been included in previous studies21 (link),23 (link),28 (link),84 (link).
8
Subcutaneous Implantation and Cell Analysis
9
Isolation and Cryopreservation of Chondrocytes
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OCD cartilage fragments digested in 2 mg/mL collagenase solution (Worthington Biochemical Corp. 290 active units/mg DW) supplemented with 30 µL/mL (3% v/v) fetal bovine serum (FBS) (Atlanta Biologicals) for 18 hr. at 37°C, 5% CO2 with orbital rotation at 60 RPM. HAC-chondrocytes were obtained from the articular surface of the humeral head by transverse scoring of the articular surface followed by horizontal scoring and processed as described above for OCD cartilage. A cell count and viability were obtained using a hemocytometer for all specimens using Trypan blue vital staining. Chondrocytes that were not immediately used for expansion were cryopreserved in liquid nitrogen in 90% FBS and 10% Dimethyl Sulfoxide (DMSO) (Corning® DMSO, Mediatech Inc.) until further use.
10
Isolation and Culture of Primary HUVECs
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Primary HUVECs were isolated as previously described [22 (link),23 (link)]. In brief, the umbilical cord vein from normal term placentas was cannulated, and fetal blood was washed out. Pre-warmed 1 mg/mL collagenase solution (Worthington, Lakewood, NJ, USA) was infused into the cord, followed by incubation at 37 °C for 8 min to allow HUVECs to dissociate. HUVECs were recovered by pelleting and resuspension, followed by culture, and used between passages 1 and 3. Cells were seeded for treatment in M199 media (Life Technologies, Carlsbad, CA, USA) containing 10% fetal calf serum (Sigma-Aldrich, St Louis, MO, USA), 1% endothelial cell growth factor, 1% heparin (Sigma), and 1% Antibiotic-Antimycotic (AA, ThermoFisher Scientific, Waltham, MA, USA) and incubated under 20% O2, 5% CO2 at 37 °C overnight.
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