An adult Aplysia californica (~100 g) was anesthetized by injecting a half body weight of isotonic MgCl2 solution (0.39 M). The abdominal ganglion was dissected and treated with protease XIV from Streptomyces griseus (Sigma-Aldrich) (10 mg/mL in ASW) at 34 oC for 90 min. Bag cells were then manually isolated from the ganglion and loaded into polyimide capillary as described above.
Protease xiv from streptomyces griseus
Manufactured by Merck Group
Sourced in United States
Protease XIV from Streptomyces griseus is a laboratory enzyme used for protein digestion. It is a proteolytic enzyme that can hydrolyze peptide bonds in various proteins.
Automatically generated - may contain errors
Lab products found in correlation
Sodium chloride (nacl), by Merck Group (2 mentions)
Potassium chloride (kcl), by Merck Group (2 mentions)
Taurine, by Merck Group (2 mentions)
Mgso4, by Merck Group (2 mentions)
Kolliphor hs 15, by Merck Group (1 mentions)
Phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Fatty acid free bsa, by Thermo Fisher Scientific (1 mentions)
Pf543, by Cayman Chemical (1 mentions)
Collagenase, by Worthington (1 mentions)
Trypan blue solution, by Thermo Fisher Scientific (1 mentions)
Tunicamycin, by Merck Group (1 mentions)
Nahco3, by Merck Group (1 mentions)
Cacl2, by Merck Group (1 mentions)
Triphenyltetrazolium chloride, by Merck Group (1 mentions)
Collagenase 2, by Worthington (1 mentions)
2 3 butanedione monoxime, by Merck Group (1 mentions)
Pd98059, by Cell Signaling Technology (1 mentions)
7 protocols using protease xiv from streptomyces griseus
1
Isolation of Aplysia Bag Cells
2
Cardiomyocyte Isolation Protocol
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Reagents used in cardiomyocyte isolation (NaCl, KCl, MgSO4, CaCl2, NaHCO3, 2,3-Butanedione Monoxime, glucose, protease XIV from streptomyces griseus and taurine), were purchased from Sigma-Aldrich (St. Louis, MO). The collagenase II component of the digestion buffer was purchased from Worthington Biochemical Corporation (Lakewood, NJ). D17 Sphingosine for the sphingosine kinase (SK) assay was purchased from Avanti Polar Lipids (Birmingham, AL), and phosphatase inhibitors and fatty acid free BSA were obtained from Fisher scientific. Recombinant relaxin (serelaxin) was provided by Novartis pharmaceuticals (Basel, Switzerland). PF543 was obtained from Cayman Chemicals (Ann Arbor, MI). Kolliphor (HS-15) was obtained from Sigma-Aldrich.
3
Zein-based Protease Enzyme Delivery
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Zein was procured from WAKO Pure Chemical Industries Ltd., Japan. Protease XIV from Streptomyces griseus (3.5 U/mg) was purchased from Sigma-Aldrich, USA, and Ciprofloxacin HCl (CPFX) was purchased form Shanghai Macklin Biochemical Co., Ltd., China. Doxorubicin hydrochloride (DOX) was obtained from Aladdin, China. Carbopol 980 was purchased from Lubrizol Co., USA.
4
Silk Inverse Opal Protease Degradation
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Proteinase K from Tritirachim
album (Sigma), protease XIV from Streptomyces griseus (Sigma), and collagenase (Worthington) stock solutions were prepared
to 400, 50, and 1500 U mL–1, respectively. For time-lapse
protease degradation experiments, 20 μL of proteinase K stock
solution was added at a location close to the silk inverse opal and
imaged using a CCD camera. Working protease concentrations (Proteinase
K: 4 U mL–1, protease XIV: 1× = 2 U mL–1, and collagenase: 1× = 150 U mL–1) were applied to silk inverse opals, and opal spectra were analyzed
over time using the spectrophotometer.
album (Sigma), protease XIV from Streptomyces griseus (Sigma), and collagenase (Worthington) stock solutions were prepared
to 400, 50, and 1500 U mL–1, respectively. For time-lapse
protease degradation experiments, 20 μL of proteinase K stock
solution was added at a location close to the silk inverse opal and
imaged using a CCD camera. Working protease concentrations (Proteinase
K: 4 U mL–1, protease XIV: 1× = 2 U mL–1, and collagenase: 1× = 150 U mL–1) were applied to silk inverse opals, and opal spectra were analyzed
over time using the spectrophotometer.
5
Silk Scaffold Enzymatic Degradation
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Silk scaffolds (n = 15) were exposed to protease XIV from Streptomyces griseus (Sigma–Aldrich, St. Louis, MO, USA) to monitor and compare enzymatic degradation profiles over the course of 21 days, as an in vitro model of scaffold degradation [47 (link),48 (link),49 (link)]. After recording the initial scaffold mass dry, scaffolds were plated in a 48-well plate and each submerged in 500 µL of 181 µg/mL (1 U/mL) protease XIV solution (prepared in sterile distilled water, with approximately 2% penicillin/streptomycin), with continuous incubation at 37 °C. The protease solution was changed daily for each scaffold. On three-day intervals, scaffolds were taken out of enzyme solution and rinsed 3 times in sterile distilled water before drying (8 h at 60 °C) to monitor changes in dry mass across measurement timepoints. Changes in dry mass for each scaffold were normalized to initial scaffold mass to compare percent reduction over the course of the study. Discarded protease solution was denatured by incubation for 20 min at 80 °C according to the manufacturer’s protocol.
6
Isolation and Culture of Myocytes
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Triphenyltetrazolium chloride and tunicamycin were purchased from Sigma‐Aldrich. Trypan blue solution (0.4%) was obtained from Thermo Fisher Scientific. PD98059 (Erk 1/2 inhibitor) was purchased from Cell Signaling Technologies. Phthalo blue dye was purchased from Quantum Ink (Louisville, KY). B7‐33 was kindly provided by our collaborators from University of Melbourne (Victoria, Australia). Reagents used in myocyte perfusion and digestion buffers (NaCl, KCl, MgSO4, CaCl2, NaHCO3, 2,3‐butanedione monoxime, glucose, protease XIV from streptomyces griseus, and taurine) were purchased from Sigma‐Aldrich. Collagenase II was obtained from Worthington Biochemical Corporation (Lakewood, NJ).
7
Enzymatic Degradation of Silk Fibroin Membranes
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Membranes were tested using an accelerated in vitro enzymatic degradation assay with protease XIV from Streptomyces griseus (Sigma-Aldrich) using a modified method based on previous work [20]. Briefly, dry SF membrane samples were weighed and treated with 1 mg/mL protease solution (equal to 3.5 U/mL) dissolved in 0.1 M phosphate saline buffer (PBS) at pH 7.4, in ratio 1:150 w/w. Controls were incubated in PBS alone. The membranes were incubated at 37°C at 50 rpm in a shaker. Samples were collected at different times (0, 1, 2, 4, 6, 12, 24 hours) and analysed using optical coherence tomography (OCT), which provides volumetric imaging of the sample microstructure based on its optical back-scattering properties.
Volumetric OCT scans were acquired using a fiber-based spectral-domain OCT system (Telesto 320, Thorlabs Inc., USA). The light source is a superluminescent diode with a mean wavelength of 1300 nm and a spectral bandwidth of 170 nm. The measured axial and lateral resolution (in air) is 4.8 µm and 7.2 µm respectively. Scans were acquired by taking 1000 by 1000 axial line scans across a 10 mm by 10 mm lateral field of view. For each sample, the SF thickness was determined from the OCT scan using morphological image processing.
Volumetric OCT scans were acquired using a fiber-based spectral-domain OCT system (Telesto 320, Thorlabs Inc., USA). The light source is a superluminescent diode with a mean wavelength of 1300 nm and a spectral bandwidth of 170 nm. The measured axial and lateral resolution (in air) is 4.8 µm and 7.2 µm respectively. Scans were acquired by taking 1000 by 1000 axial line scans across a 10 mm by 10 mm lateral field of view. For each sample, the SF thickness was determined from the OCT scan using morphological image processing.
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