Collagenase-induced OA (CiOA) was induced according to our current standard protocol, as previously published [27 (link)]. On day zero and day two of CiOA, mice received an intra-articular injection of 1U bacterial collagenase type VII (Sigma-Aldrich, St. Louis, MO, USA) into the right knee cavity. Investigators were aware of diet groups due to bright green food coloring in the WD, but blinded for genotype. On day 7 and day 21 of CiOA, mice were weighed, blood was collected via orbital plexus bleeding and mice were sacrificed, after which right knee joints were isolated and processed for histological analysis. On day seven of CiOA, the synovia were dissected from the knees before histological processing, weighed, and incubated for 2 h in DMEM (0.5% BSA) to obtain conditioned media. Synovial punches for qPCR analyses were obtained from a different experiment in which the identical diet was fed to C57Bl/6 mice starting four weeks prior to CiOA induction [27 (link)]. Serum samples were obtained from all mice to determine lipoprotein, S100A8/A9 and cytokine concentrations.
Bacterial collagenase type 7
Manufactured by Merck Group
Sourced in United States
Bacterial collagenase type VII is a laboratory enzyme used to break down collagen, a structural protein found in various tissues. It is derived from the bacterium Clostridium histolyticum. The enzyme is used in research and scientific applications that require the digestion or dissociation of collagen-containing samples, such as tissue culture and cell isolation procedures.
Automatically generated - may contain errors
10 protocols using bacterial collagenase type 7
1
Collagenase-Induced Osteoarthritis Model
2
Rat Model of Striatal Collagenase Injection
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The ICH rat model was used as previously described.21 Anesthesia was maintained by inhalation of 4% sevoflurane in a N2O/O2 mixture (70/30), and body temperature was maintained at 37±0.5°C with a heating pad until animals completely recovered from anesthesia and displayed normal motor activity. Glucose levels were analyzed before surgery (mean 144.3±22.1 mg/dL). Rats were placed in a stereotaxic frame (Stoelting Co, Wood Dale, IL) under sevoflurane anesthesia. After drilling of a small burr hole, 1 μL of saline containing 0.2 U/μL bacterial collagenase type VII (Sigma‐Aldrich Corp, St. Louis, MO) was injected into the right striatum (0.6 mm anterior to bregma, −3.0 mm lateral and 5.5 mm depth) using a Hamilton syringe with a 30 G needle. Injections took place over a 10‐minute period, and the needle remained in place for additional 10 minutes before removal (1 mm/min). The burr hole was filled with bone wax (Ethicon, Somerville, NJ), and the scalp incision was closed with sutures.
3
Collagenase Enzymatic Activity Assay
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Enzymatic assay of collagenase was performed using FALGPA (Sigma‐Aldrich Corp, St. Louis, MO) as substrate, according to the manufacturer's recommendations. Briefly, proteolysis of 1 mmol/L FALGPA by 2 U/mL bacterial collagenase type VII (Sigma‐Aldrich Corp, St. Louis, MO) in reaction buffer (50 mmol/L Tricine; 10 mmol/L CaCl2; 400 mmol/L NaCl) was monitored spectrophotometrically (345 nm) for 5 minutes, in the presence of CM352, phenanthroline, or vehicle.
4
Extraction and Fractionation of Bovine Corneal Collagen VI Microfibrils
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Collagen VI microfibrils were extracted from bovine cornea using collagenase as described previously [34] (link). Approximately 0.2 g (wet weight) of bovine cornea was diced before being suspended in 2 ml of digestion buffer (400 mM NaCl, 20 mM Tris-HCl (pH 7.4)) which contained 0.1 mg/ml of chromatographically purified bacterial collagenase type VII (Sigma-Aldrich), and protease inhibitors (3 mM NEM, 5 mM PMSF). Digestions were incubated overnight at 4 °C whilst undergoing gentle stirring. Digested tissue was centrifuged for 3 min at 800g and the supernatant size fractionated on a Sepharose CL-2B column (GE Healthcare) equilibrated in 150 mM NaCl, 20 mM Tris-HCl, 2.5 mM CaCl2 (pH 7.4).
5
Intracerebral Collagenase-Induced Intracerebral Hemorrhage Model
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The ICH model was induced by stereotactic administration of 0.5 U bacterial collagenase type VII (Sigma Aldrich, USA) as described previously (26 (link)). Rats were divided into five groups, with one control (sham), and four ICH groups receiving an intracerebral injection of saline, normal MSCs, MSCs infected with empty lentivirus (NC-MSCs), or MSCs infected with miR-21-overexpressing lentivirus (miR-21-MSCs). A total of 106 MSCs in 10 μl of saline were transplanted by intracerebral injection 3.0 mm right-lateral to the midline, 1.0 mm posterior to the bregma, 5.0 mm in depth below the skull. Neurological behavioral assessments were made on days 3, 7, and 14 after ICH, using the corner test and forelimb placement experiment as previously described (27 (link)). The wet weight of each brain was immediately obtained using an electronic balance, following which the brains were dried at 100°C for 24 h to obtain the dry weight. Water content was calculated as previously describe (28 (link)).
6
Intracerebral Hemorrhage Induction and GHK Treatment
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Intracerebral hemorrhage was induced via stereotactic administration of 0.25 U/μl bacterial collagenase type VII (Sigma, United States), as Rosenberg described (Rosenberg et al., 1990 (link)). Rats in the GHK treatment group were intraperitoneally injected with GHK every 24 h dissolved in saline for 3 days after the administration of ICH, while those in the control group were treated with equal volumes of vehicle.
7
Stereotaxic Collagenase Injection in Rat Striatum
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Rats were anesthetized using an intramuscular (i.m.) injection of 1% zoletil (20 mg/kg) and xylazine hydrochloride (5 mg/kg). In brief, 1 µl (0.2 µl/min) saline containing 0.2 units of bacterial collagenase (type VII; Sigma) was injected stereotaxically into the striatum at coordinates of 3.0 mm lateral to bregma and 5.0 mm ventral to the cortical surface. After injection, the Hamilton syringe was left in place for 5 minutes. The needle was slowly removed after an additional 10 minutes to prevent backflow. The hole was sealed with bone wax and the wound was sutured.
8
Stereotactic Collagenase-Induced ICH
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ICH was induced via stereotactic administration of 0.5 U bacterial collagenase type VII (Sigma, United States) in 2 μl saline, as previously described (Rosenberg et al., 1990 (link)).
9
Collagenase-Induced Intracerebral Hemorrhage in Mice
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ICH mouse model was prepared based on the previous literature [30 (link)]. Mice were anesthetized with pentobarbital (40 mg/kg) by intraperitoneally (ip) injection. Thereafter, mice were positioned on a stereotactic frame (RWD, Shenzhen, China). An incision was made in the scalp and the skull was exposed extensively. A small hole was drilled on the skull’s right side of (coordinates: 2.0 mm lateral to midline, 0.8 mm anterior to the bregma). Bacterial collagenase type VII (0.075 units, Sigma-Aldrich, Milwaukee, WI, USA) was dissolved in 0.5 μL of sterile saline and infused into brain at a rate of 0.1 μL/min with an infusion pump (3.5 mm deep relative to bregma). After injection, the needle was left in the same position for 10 min to prevent reflux and then slowly withdrawn. The cranial burr hole was covered with bone wax, and the wounded scalp was sutured. Sham group were injected with an equal volume of sterile saline.
10
Collagenase-Induced Rat Intracerebral Hemorrhage
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Intrastriatal injection of collagenase type VII was used to induce the rat ICH model as previously described with modifications (13 (link)). An intraperitoneal injection of 10% chloral hydrate (3 mL/kg) was used to anesthetize the rats. Then the rats were placed in a stereotaxic frame (Narishige SN-3, Tokyo, Japan) under aseptic conditions. After bregma was exposed, a 30-G needle was inserted into the striatum through a burr hole that was 0.2 mm posterior and 3.0 mm lateral to bregma, and 6.0 mm below the surface of the skull. A saline solution (3 µL) containing 0.8 U of bacterial collagenase type VII (Sigma-Aldrich, St. Louis, MO, USA) was injected over a duration of 5 min. The needle was withdrawn slowly after an additional 5 min. Bone wax was used to seal the burr hole. The scalp was then sutured. The sham-operated rats were treated simultaneously with the same method except for injection of an equivalent volume of sterile saline instead of collagenase. All rats survived the operation.
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