Mouse smooth muscle cells were isolated as described before (Lauer and others 2009a (link), 2009b (link)). Briefly, tracheas were excised, longitudinally cut, and then digested in 0.15% Pronase solution (Roche) at 4°C overnight. Next, the remaining epithelial cells were removed with a cotton swab and tracheas were cut into small pieces (∼30 per trachea). Trachea fragments were transferred to a 100-cm2 tissue culture dish for attachment and outgrowth of ASMCs.
Pronase solution
Manufactured by Roche
Sourced in Switzerland
Pronase solution is a proteolytic enzyme preparation derived from the bacterium Streptomyces griseus. It is commonly used in a variety of laboratory applications to break down or degrade protein-based samples.
Automatically generated - may contain errors
Lab products found in correlation
Collagenase b solution, by Roche (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Protease inhibitor, by Roche (1 mentions)
Collagenase, by Thermo Fisher Scientific (1 mentions)
Hyaluronidase solution, by Merck Group (1 mentions)
Dmem high glucose, by Merck Group (1 mentions)
Hepes, by Merck Group (1 mentions)
L ascorbate 2 phosphate, by Merck Group (1 mentions)
Insulin, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
Papain, by Merck Group (1 mentions)
Antibiotic antimycotic solution, by Merck Group (1 mentions)
Collagenase solution, by Roche (1 mentions)
7 protocols using pronase solution
1
Isolation of Mouse Smooth Muscle Cells
2
Cultivation of Immortalized and Primary Chondrocytes
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Human immortalized articular chondrocyte C28/I2 cells were purchased from Merck Millipore and cultured in DMEM/F12 (Gibco) containing 10% FBS (Gibco), 1% (vol/vol) antibiotic/antimycotic (Gibco), and 1% L-glutamine (Gibco) in a humidified atmosphere at 37°C and 5 % CO2. For the isolation of primary hACs, cartilage was first dissected from the joint surface, rinsed with PBS, and cut into small pieces. The cartilage pieces were incubated with 2 mg/ml pronase solution (Roche) for 90 minutes at 37°C and digested overnight at 37°C in 1.5 mg/ml collagenase B solution (Roche). Then, the preparation was filtered through a 70 μM strainer and cells were plated in culture flasks and cultured in a humidified atmosphere at 37°C and 5% CO2. Culture medium consisted of DMEM/F12 (Gibco), 10% FBS (Gibco), 1% (vol/vol) antibiotic/antimycotic (Gibco), and 1% L-glutamine (Gibco).
3
Protein Extraction and Protease Digestion
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Extraction of BMMs cell protein was obtained using mammalian protein extraction lysis buffer (Thermo Fisher Scientific) with addition of 1% protease inhibitor (Roche). A bicinchoninic acid (BCA) protein concentration assay was performed to determine protein concentration of cell lysate. Aliquots of 50 μg of protein from the lysate were taken for each sample, and then oroxylin A was added to samples to final concentrations of 0, 0.05, 0.25, and 0.50 mM, followed by incubation for 60 min at room temperature. After incubation, pronase solution (Roche) was added to samples [pronase:protein = 1:1000, (w/w)] for digestion for 30 min at room temperature. We ensured that each sample was digested for the same amount of time. Samples without addition of pronase were prepared as controls. The digestion reaction was stopped by adding 5× SDS–polyacrylamide gel electrophoresis loading buffer, followed by heating at 95°C for 10 min. Western blotting analyses were performed to examine the degradation levels of LPL in each sample.
4
Isolation and Expansion of Human Articular Chondrocytes
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Human articular chondrocytes were collected from the femoral head of three patients undergoing hip replacement, with patient’s consent and the approval of the ethical board of the Medical University of Vienna (No 1741/2018). Articular chondrocytes were isolated by 1 h incubation in 0.1% hyaluronidase solution (Sigma-Aldrich, Austria), 0.5 h in 0.1% pronase solution (Roche, Switzerland) followed by overnight incubation in a mixture of 200 U/mL collagenase (Gibco, United States) and 1 U/mL papain (Sigma-Aldrich, Austria). HAC were expanded in monolayer culture until passage two prior to hydrogel embedding. Medium was exchanged twice a week using chondrogenic expansion medium consisting of DMEM high glucose with 10% FCS, 1% antibiotic antimycotic solution, 10 mM HEPES, 6 mM L -glutamine, 5 μg/mL insulin, 50 μg/mL L -ascorbate-2-phosphate (all from Sigma-Aldrich, Austria).
5
Isolation of Chondrocytes from Human Cartilage
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Human articular cartilage as part of surgical waste obtained from three donors without osteoarthritis was provided by the University of Wisconsin Hospital and Clinics with approval from the Institutional Review Board. To isolate chondrocytes, cartilage was individually minced, washed twice with D-PBS, and incubated with pronase solution (1 mg/ml; Roche, Basel, Switzerland) for 30 min at 37°C and additionally with collagenase solution (1 mg/ml; Roche) for 12 to 18 hours at 37°C with agitation. The digestion solution was collected and centrifuged at 600g for 5 min to prepare uncultured NCs for RNA-seq analysis.
6
Isolation and Culture of Mouse Airway Smooth Muscle Cells
7
Chondrogenic Potential of iPSC-derived Chondrocytes
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Adult human articular chondrocytes (hAC) were used to compare the chondrogenic phenotype of iPSC derived chondrocytes. The hACs were obtained from patients undergoing hip replacement for osteoporotic or malignancy-associated fractures (female: 82, male: 54 and male 84 years old). The University Hospitals Leuven Ethics Committee and Biobank Committee approved the study. For chondrocytes isolation, cartilage was dissected from the joint explant surfaces and then rinsed with saline. The tissue was cut into small pieces, using a sterile surgical blade. Cartilage explants were incubated with 2 mg/ml pronase solution (Roche) for 90 min at 37 • C under continuous agitation and digested overnight at 37 • C in 1.5 mg/ml collagenase B solution (Roche). The preparation was filtered through a 70 μM strainer and cells were plated in culture flasks and cultured in a humidified atmosphere at 37 • C, 5% CO 2 with a density of 16 000 cells cm -2 . Culture medium consisted of DMEM/F12 (Gibco), 10% fetal bovine serum (FBS) (Gibco), 1% (vol/ vol) antibiotic/antimycotic (Gibco) and 1% L-glutamine (Gibco). Experiments were performed with passage 2 cells in triplicate.
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