Referring to our previous study [10 (link)], AF samples harvested from fresh bovine tails were frozen in liquid nitrogen for 22 h prior to thawing in a hypotonic buffer (10 mM tris–HCl, pH 8.0) at 37 °C for 2 h. A 75% ethanol pre-wash of AF samples was then conducted for 2 h. Subsequently, AF samples were placed in the decellularization solution, which consisted of tris–HCl buffer (10 mM Tris–HCl, pH 8.0) containing 0.2% SDS, 0.1% EDTA, and 10 KIU/mL aprotinin at 4 °C for 24 h with gentle agitation (40 r/m) on an orbital shaker. After transient rinsing, samples were submerged in deoxyribonuclease (DNase 50 U/mL; Sigma, USA) and ribonuclease (RNase 1 U/mL; Sigma, USA) in Tris buffer (50 mM tris–HCl, 10 mM magnesium chloride, and 50 mg/mL bovine serum albumin at pH 7.5) for 3 h at 37 °C with gentle agitation. Decellularized AF scaffolds were eventually acquired after being constantly washed three times in PBS for 8 h. Scaffolds were freeze-dried and stored in a sterile environment for further use.
Deoxyribonuclease dnase
Manufactured by Merck Group
Sourced in United States, Germany
Deoxyribonuclease (DNase) is an enzyme that catalyzes the hydrolytic cleavage of DNA molecules. It functions by breaking down the phosphodiester bonds within the DNA backbone, effectively reducing the size and complexity of DNA fragments. This enzymatic activity is a core process in various laboratory procedures, such as DNA extraction, purification, and manipulation.
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Lab products found in correlation
Hepes buffer solution, by Thermo Fisher Scientific (3 mentions)
Collagenase, by Worthington (3 mentions)
Antibiotic antimycotic mixture, by Thermo Fisher Scientific (2 mentions)
Poly l lysine (pll), by Merck Group (2 mentions)
Collagenase, by Merck Group (2 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (2 mentions)
Trypsin, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Hanks balanced salt solution (hbss), by Worthington (1 mentions)
Facs fortessa, by BD (1 mentions)
Liberase tl, by Roche (1 mentions)
Fixation and permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Phorbol 12 myristate 13 acetate, by Merck Group (1 mentions)
D glucose, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Papain dissociation solution, by Worthington (1 mentions)
Dl cysteine, by Merck Group (1 mentions)
Papain, by Worthington (1 mentions)
70 m nylon mesh, by Celltreat (1 mentions)
20 protocols using deoxyribonuclease dnase
1
Decellularization of Bovine Articular Cartilage
2
Primary Leiomyoma and Myometrial Cell Isolation
3
Tumor Digestion and Immune Cell Analysis
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Mice were euthanized around 15 days after transplantation when the diameter of the tumor reached 8 to 10 mm. Isolated tumors were cut into small pieces and digested with Liberase TL (0.25 mg/ml) (Roche) and deoxyribonuclease (DNase) (0.33 mg/ml) (Sigma-Aldrich) in 37°C for 30 min. Until now, single-cell suspensions were obtained. For IFN-γ and granzyme B staining, cells were stimulated for 4 hours with phorbol 12-myristate 13-acetate (50 ng/ml) (Sigma-Aldrich) and ionomycin (1 μg/ml) (Sigma-Aldrich) in the presence of brefeldin A (10 μg/ml). After stimulation, cells were stained for indicated antibodies of surface marker, followed by treatment with fixation and permeabilization buffer (eBioscience) according to the manufacturer’s instructions. Cells were further stained with antibodies of intracellular markers. All the samples were applied to Fortessa FACS (BD Biosciences) and analyzed by Flowjo software (TreeStar).
4
Isolation and Culture of Mouse Hippocampal Neurons
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Mice primary hippocampal neuronal cultures were prepared as previously described by others 57 (link). Except when otherwise indicated, all reagents were from Gibco. Briefly, hippocampi were dissected from P2 mouse pups in cold Hanks’ balanced salt solution (HBSS) supplemented with 0.08% D-glucose (Sigma-Aldrich), 0.17% Hepes, and 1% penicillin-streptomycin (Pen-Strep); filter-sterilized; and adjusted to pH 7.3. After dissection, the hippocampi were washed twice with cold HBSS and individually incubated at 37°C for 20 min in papain dissociation solution (45 U of papain (Worthington), 0.01% deoxyribonuclease (DNase), 1 mg of DL-cysteine, 1 mg of bovine serum albumin (BSA) and 25 mg of D-glucose (all from Sigma-Aldrich) in phosphate-buffered saline (PBS). After digestion, the hippocampi were washed twice with DMEM preheated to 37°C and supplemented with 10% FBS and dissociated by 10 cycles of aspiration through a micropipette tip. Dissociated neurons were then resuspended in warm DMEM supplemented with 10% FBS and plated in 6-well plates containing 25-mm sonicated glass coverslips pretreated with 50 μg/ml poly-L-lysine (PLL, Sigma-Aldrich). After 1 hour, the medium was replaced with Neurobasal-A medium, which was supplemented with 2% B-27 and 0.25% GlutaMAX (neuronal medium). Primary neurons were maintained in a standard tissue culture incubator at 37°C with 5.5% CO2.
5
Isolation and Analysis of Tumor-Infiltrating Lymphocytes
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Blood samples were drawn from CT26 tumor-bearing mice on day 7 post-treatment. For each sample, 25 µL of fresh heparinized blood was incubated with the appropriate dilution of fluorescence-conjugated antibodies (see Flow cytometry section) for 30 min at 4°C in the dark. Tumors were harvested 7 days post-treatment, cut into small fragments, and digested in 1 mg/mL collagenase and 20 mg/mL deoxyribonuclease (DNase) (Sigma) in phosphate buffered saline for 45 min at room temperature. Subsequently, tumor-infiltrating lymphocytes (TILs) were filtered through 70 µm nylon mesh (Cell Treat), washed with 10 mL complete Roswell Park Memorial Institute media (RPMI), and collected by centrifugation (1500 rpm, 4 min). Pelleted cells were resuspended for staining and analysis by flow cytometry (see Flow cytometry section).
6
Western Blot Analysis of Cell Signaling
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Lysis of the cell pellets was performed in 50 mM tris (pH 8.0), 150 mM NaCl, 0.5% NP-40, 50 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride (PMSF), one tablet protease inhibitors (EDTA free, Roche) per 10 ml, and deoxyribonuclease (DNase; 30 μg/ml) (Sigma-Aldrich). After Western blotting and wet transfer in ethanol-based transfer buffer onto nitrocellulose membranes (Cytiva), proteins were detected by chemoluminescence (Advansta, K-12049-D50) using rat anti–caspase-2 (11B4, Enzo Life Science), rabbit anti-p19ARF (sc-32748, Santa Cruz Biotechnology), mouse anti-p53 (1C12, Cell Signaling), rabbit anti-p21 (ab7960), mouse anti-HSP90 (sc-13119, Santa Cruz Biotechnology), rabbit anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (14C10, Cell Signaling), and mouse anti-hBCL2 (clone S100). Goat anti-rabbit Ig/horseradish peroxidase (HRP) (Dako, P0448) or rabbit anti-mouse Ig/HRP (Dako, P0161) was used as secondary reagent, respectively.
7
Decellularization of Cartilage Scaffold
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The protocol started with an initial dry 12 hour freeze thaw of the specimens followed by thawing at room temperature. This was followed by another 12 hour wet freeze thaw cycle in phosphate buffer solution (PBS, Sigma-Aldrich, USA) at −20 °C. The addition of a wet freeze thaw cycle in PBS allowed for cellular remnants to be washed away from the scaffold after thawing at room temperature. In addition it exerted greater mechanical pressure on the cartilage in physically decellularizing it. The cartilage was subsequently washed in deionized water overnight under agitation at room temperature. The next step involved transferring the tissue specimens to 4% sodium deoxycholate solution (Sigma-Aldrich, USA) under agitation at room temperature for four hours. This was subsequently followed by a wash in PBS solution for 30 minutes under agitation at room temperature. Next, the tissue specimens were submersed in 2% deoxyribonuclease/DNase (Sigma-Aldrich, USA) for 3 hours. This was followed by an overnight wash in deionized water with agitation at room temperature. The process was repeated for subsequent cycles however the initial freeze thaw steps were only performed once at the beginning of the protocol (Fig. 2 ).![]()
Summary of steps for protocol A, B and C.
8
Cell Culture Reagents and Hormones
9
Murine Uterine and Placental Cell Isolation
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On GD 14, the mouse uterus and placenta were carefully separated and washed two to three times with cold PBS. The tissues were cut into 1- to 3-mm fragments with ophthalmic scissors. Then, 1 mg/ml collagenase IV (Biofroxx, Germany) and 0.2 mg/ml deoxyribonuclease (DNase; Sigma–Aldrich, St. Louis, Mo) were added, and the samples digested in a biochemical incubator at 37 ℃ for 45 min. The digested tissue was then filtered through 48-μm sterile nets and washed twice in cold PBS. Finally, the cells were collected and resuspended in cold PBS. Mononuclear cells were obtained by aspirating the white membrane layer in the mouse lymphocyte isolation medium (TBD Science, China) after Ficoll density gradient centrifugation, and were used for flow cytometry.
10
Isolation and Culture of Mesenchymal Stem Cells
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MSCs were isolated as previously described [19 (link)]. Briefly, cells were isolated from patients undergoing hysterectomy at the Johns Hopkins University Hospital. The Institutional Review Board of the Johns Hopkins University reviewed and approved the study (IRB00196175), and informed consent was obtained from all patients to participate. Tissue was transferred to the laboratory immediately after surgery and washed several times with a Hanks’ Balanced Salt Solution (HBSS, Thermo Fisher Scientific, Waltham, MA, USA) without calcium or magnesium. Tissue was then manually cut into 1–2 mm3 specimens and incubated in sterile HBSS (without calcium, or magnesium) with collagenase (Worthington, Lakewood, NJ, USA), deoxyribonuclease (DNase, Sigma-Aldrich, St. Louis, MO, USA), antibiotic–antimycotic mixture (Thermo Fisher Scientific), and HEPES buffer solution (Thermo Fisher Scientific) at 37 °C on a shaker for 4–8 h. The digest was filtered through a 100 µm and then a 40 µm filter and cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F12 (DMEM/F-12) (Thermo Fisher Scientific) medium supplemented with HEPES, L-glutamine, 10% FBS, and 1% antibiotic–antimycotic.
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