Synovium was digested with 2 mg/mL collagenase (Wako), 3 mg/mL dispase (Wako), and 25 μg/mL deoxyribonuclease I (Sigma-Aldrich) prepared in Dulbecco’s Modified Eagle’s Medium (DMEM, Life Technologies) with shaking at 37°C for 1 hour. Bone fragments were crushed with a pestle, after which the crushed bones were washed gently once in phosphate buffered saline (PBS) (for remove the marrow cells). Bone and bone fragments were incubated for 1 hour at 37°C in DMEM in the presence of 2 mg/mL collagenase (Wako Chemicals) and 25 μg/mL deoxyribonuclease I (Sigma-Aldrich). The cell suspensions (synovium and bone fragments) were filtered through a cell strainer (Falcon, 70 μm) to remove debris. After lyse the red blood cells, these cells were re-suspended in calcium- and magnesium-free Hank's Balanced Salt Solution (HBSS) (Gibco) supplemented with 2% FBS, 10 mM HEPES and 1% penicillin/streptomycin.
Deoxyribonuclease 1
Manufactured by Merck Group
Sourced in United States, United Kingdom, Japan, Italy
Deoxyribonuclease I is an enzyme that catalyzes the hydrolytic cleavage of DNA. It is a common laboratory reagent used to degrade DNA in various experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (19 mentions)
Collagenase, by Merck Group (15 mentions)
Trypsin, by Merck Group (14 mentions)
Collagenase 4, by Merck Group (10 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (10 mentions)
Percoll, by Merck Group (9 mentions)
L glutamine, by Thermo Fisher Scientific (9 mentions)
Streptomycin, by Thermo Fisher Scientific (9 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (8 mentions)
Hyaluronidase, by Merck Group (7 mentions)
Epiquik m6a rna methylation quantification kit, by Epigentek (7 mentions)
Penicillin, by Thermo Fisher Scientific (7 mentions)
Ribonuclease a, by Merck Group (6 mentions)
Trizol reagent, by Thermo Fisher Scientific (6 mentions)
Trypsin, by Thermo Fisher Scientific (6 mentions)
Poly d lysine, by Merck Group (5 mentions)
Hyaluronidase type 5, by Merck Group (5 mentions)
Falcon cell strainer, by BD (5 mentions)
Liberase, by Merck Group (5 mentions)
Neurobasal medium, by Thermo Fisher Scientific (5 mentions)
174 protocols using deoxyribonuclease 1
1
Isolation of Synovial and Bone Cells
2
Isolation of Endometrial and Blood Cells
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Endometrial samples were obtained using an Endocell disposable endometrial cell sampler (Wallach Surgical devices, CT, USA) under ultrasound guidance starting from the uterine fundus and moving downwards to the internal cervical os.
Samples were washed in PBS to remove blood contamination and tissue cut into 2 mm3 fragments before incubation with 1x Liberase (Roche Life Sciences) and 100 μg/ml Deoxyribonuclease 1 (Sigma Aldrich) for 45 minutes at 37 °C with shaking. Digested tissue was resuspended in 10 ml ‘R10’ growth media [Iscove’s Modified Dulbecco’s Medium (Sigma Aldrich), 10% Fetal Calf Serum (FCS) (Gibco), 2% Human Serum (Sera Laboratories International), 1% penicillin-streptomycin (50 IU/ml and 50 μg/ml), 1% glutamine] and filtered through consecutive 70 μm and 40 μm mesh cell strainers (BD Falcon) then cells were washed by centrifugation at 600 × g for 5 minutes.
10 ml peripheral blood was collected into sodium heparin anti-coagulant (10 U/ml) and peripheral blood mononuclear cells (PBMC) isolated using lymphoprep (Axis Shield Diagnostics). PBMC were washed twice in PBS/1% FCS with centrifugation at 800 × g and 200 × g for 10 minutes, then used immediately for flow cytometry.
Samples were washed in PBS to remove blood contamination and tissue cut into 2 mm3 fragments before incubation with 1x Liberase (Roche Life Sciences) and 100 μg/ml Deoxyribonuclease 1 (Sigma Aldrich) for 45 minutes at 37 °C with shaking. Digested tissue was resuspended in 10 ml ‘R10’ growth media [Iscove’s Modified Dulbecco’s Medium (Sigma Aldrich), 10% Fetal Calf Serum (FCS) (Gibco), 2% Human Serum (Sera Laboratories International), 1% penicillin-streptomycin (50 IU/ml and 50 μg/ml), 1% glutamine] and filtered through consecutive 70 μm and 40 μm mesh cell strainers (BD Falcon) then cells were washed by centrifugation at 600 × g for 5 minutes.
10 ml peripheral blood was collected into sodium heparin anti-coagulant (10 U/ml) and peripheral blood mononuclear cells (PBMC) isolated using lymphoprep (Axis Shield Diagnostics). PBMC were washed twice in PBS/1% FCS with centrifugation at 800 × g and 200 × g for 10 minutes, then used immediately for flow cytometry.
3
Isolation and Culture of Rheumatoid Arthritis Synovial Cells
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A total of 6 patients with RA diagnosed based on the revised 1987 American College of Rheumatology criteria for RA were recruited. Synovial tissues were collected from 6 patients who underwent arthroplastic joint surgery and synovectomy.
Synovia obtained from RA patients were aseptically dissected from the surrounding tissues, minced, and enzymatically digested with 2 mg/mL Clostridium collagenase (Wako Pure Chemical Industries, Ltd, Osaka, Japan) and 5-10 μg/mL deoxyribonuclease 1 (Sigma Chemical Co., St. Louis, MO, USA) for 2-3 h. After digestion, the resulting single cell suspension was washed, filtered through sterile gauze and nylon mesh, washed thoroughly again, and finally resuspended in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum. The cells were then cultured overnight to allow the cells to adhere to the culture plate. After washing the plate to remove nonadherent cells, the remaining adherent cells were cultured with the indicated cytokines, and adherent cells of 2-3 passages were further used in this study. The recombinant human cytokines used in this study were as follows: IL-1β, TNFα, IL-6, IL-6Rα, IL-17A, M-CSF (all BioLegend, San Diego, CA, USA), TGF-β 1 , activin A, and RANKL (all Wako Pure Chemical Industries).
Synovia obtained from RA patients were aseptically dissected from the surrounding tissues, minced, and enzymatically digested with 2 mg/mL Clostridium collagenase (Wako Pure Chemical Industries, Ltd, Osaka, Japan) and 5-10 μg/mL deoxyribonuclease 1 (Sigma Chemical Co., St. Louis, MO, USA) for 2-3 h. After digestion, the resulting single cell suspension was washed, filtered through sterile gauze and nylon mesh, washed thoroughly again, and finally resuspended in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum. The cells were then cultured overnight to allow the cells to adhere to the culture plate. After washing the plate to remove nonadherent cells, the remaining adherent cells were cultured with the indicated cytokines, and adherent cells of 2-3 passages were further used in this study. The recombinant human cytokines used in this study were as follows: IL-1β, TNFα, IL-6, IL-6Rα, IL-17A, M-CSF (all BioLegend, San Diego, CA, USA), TGF-β 1 , activin A, and RANKL (all Wako Pure Chemical Industries).
4
Protein Purification Protocol
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LB and
S.O.C media was obtained from Sigma-Aldrich.
Ampicillin sodium salt, PageRuler prestained protein ladder, 10 to
180 kDa, imidazole (99%), ethylenediaminetetraacetic (EDTA), 1-step
TMB blotting solution, and Porablot nitrocellulose membrane (0.45
μm) were obtained from ThermoFisher Scientific. Trizma base,
potassium chloride, sodium chloride, magnesium chloride, magnesium
sulfate, glucose, glycerol, calcium chloride, deoxyribonuclease 1
from bovine pancreas, nickle sulfate, hexanal (>98%), and nicotinamide
adenine dinucleotide sodium salt were purchased from Sigma-Aldrich.
S.O.C media was obtained from Sigma-Aldrich.
Ampicillin sodium salt, PageRuler prestained protein ladder, 10 to
180 kDa, imidazole (99%), ethylenediaminetetraacetic (EDTA), 1-step
TMB blotting solution, and Porablot nitrocellulose membrane (0.45
μm) were obtained from ThermoFisher Scientific. Trizma base,
potassium chloride, sodium chloride, magnesium chloride, magnesium
sulfate, glucose, glycerol, calcium chloride, deoxyribonuclease 1
from bovine pancreas, nickle sulfate, hexanal (>98%), and nicotinamide
adenine dinucleotide sodium salt were purchased from Sigma-Aldrich.
5
Isolation and Characterization of Neutrophil Secretome
6
Standardized RNA Isolation and Gene Expression Analysis
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The number of mononuclear cells isolated from spleen samples was standardized to 5 x 106 and used for RNA isolation with the use of the RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer’s protocol. Genomic DNA remaining in the samples after RNA isolation was digested with deoxyribonuclease I (Sigma Aldrich, USA). The concentration of eluted RNA was measured with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA), and the samples were stored at -80°C until further analyzed.
Reverse transcription was carried out with the High-Capacity cDNA Reverse Transcription Kit (Life Technologies, USA) according to the manufacturer’s recommendations. The concentration of RNA was standardized to 0.5 μg per sample. The relative expression of the genes encoding CD4 and CD8 T cells receptors and IFN-γ was determined by qPCR method described previously [21 (link)] with the use of Power SYBR Green PCR Master Mix kit (Life Technologies, USA) and LightCycler 96 (Roche, Switzerland).
Reverse transcription was carried out with the High-Capacity cDNA Reverse Transcription Kit (Life Technologies, USA) according to the manufacturer’s recommendations. The concentration of RNA was standardized to 0.5 μg per sample. The relative expression of the genes encoding CD4 and CD8 T cells receptors and IFN-γ was determined by qPCR method described previously [21 (link)] with the use of Power SYBR Green PCR Master Mix kit (Life Technologies, USA) and LightCycler 96 (Roche, Switzerland).
7
Isolation and Culture of Primary Human Trophoblasts
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Cultures of primary placental trophoblast cells were performed using the Kliman’s method22 (link)39 (link). Briefly, placental tissues obtained from healthy pregnancies at term were minced and dispersed with 0.25% trypsin (Invitrogen, Carlsbad, CA) and 0.1% deoxyribonuclease I (Sigma-Aldrich, St. Louis, MO). The purified cytotrophoblasts were separated by Percoll gradient centrifugation. The cells were then diluted with DMEM containing 10% fetal bovine serum (FBS), and then plated into 12-well plates at a density of 1.2 × 106/well and cultured at 37 °C in 5%CO2-95% air.
After 48 h of plating, culture medium was replaced by FBS-free DMEM containing rosiglitazone (Sigma-Aldrich), GW9662 (Sigma-Aldrich), GW1929 (Sigma-Aldrich) or 15d-PGJ2 (Sigma-Aldrich) at the indicated concentrations. The above reagents were dissolved in DMSO and then diluted by DMEM. The vehicle control was maintained by culture media containing same volume of DMSO (typically 0.01%). Each treatment was performed in triplicate for each preparation of cells. The cell viability was assessed by MTT assay, which indicated that all the treatments had no impact on the cell viability (data not shown).
After 48 h of plating, culture medium was replaced by FBS-free DMEM containing rosiglitazone (Sigma-Aldrich), GW9662 (Sigma-Aldrich), GW1929 (Sigma-Aldrich) or 15d-PGJ2 (Sigma-Aldrich) at the indicated concentrations. The above reagents were dissolved in DMSO and then diluted by DMEM. The vehicle control was maintained by culture media containing same volume of DMSO (typically 0.01%). Each treatment was performed in triplicate for each preparation of cells. The cell viability was assessed by MTT assay, which indicated that all the treatments had no impact on the cell viability (data not shown).
8
DHCT Purification and Cell Culture
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DHCT was generously provided by Professor Dequan Yu (Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China) and purified as described previously [54 (link)]. The purity was determined to be over 95% using HPLC. Penicillin, streptomycin, trypsin, soybean trypsin inhibitor, L-glutamine, fetal bovine serum (FBS), Dulbecco minimum essential medium (DMEM), and Neurobasal medium were obtained from Life Technology (Grand Island, NY, USA). Poly-D-lysine (PDL), deoxyribonuclease I, TTX, nerve growth factor (NGF), and all inorganic chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).
9
Proteomic Analysis of Cellular Stress Response
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TNF‐α, PeproTech (300‐01A); HALO‐link resin, Promega (G1913); MLN7243, Active Biochemicals (A‐1384); MLN4924, Active Biochemicals (A‐1139); H2O2, Sigma (216763); Camptothecin, Sigma (C9911); Cycloheximide, Sigma (C1988); DPQ, Calbiochem (300270); CellTiter‐Glo, Promega (G7570); Olaparib, Cambridge Biosciences (CAY10621); PARG inhibitor PDD 17273, Tocris (5952); Sodium Butyrate, Sigma (303410); GFP‐Trap_A, Chromotek (gta), PARP‐1‐Trap_A, Chromotek (xta); phosSTOP, Sigma (4906845001); cOmplete EDTA‐free protease inhibitor cocktail (Roche‐11836170001); Pepstatin A, Sigma (P5318), Bestatin hydrochloride, Sigma (B8385); Deoxyribonuclease I, Sigma (D5025); LDS Sample Buffer, Life Technologies (NP0007); Colloidal Coomassie, Expedeon (ISB1L); IPTG, Formedium (IPTG025); Micrococcal Nuclease, NEB (M0247S).
10
Isolation of Villous Trophoblasts from Human Placenta
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For in vitro studies, we isolated villous trophoblasts from healthy human term placentas by enzymatic digestion and gravitational separation as previously described by Nikitina et al.71 (link) with minor modifications. Briefly, villi-rich tissues were digested four times with 0.25% trypsin (Sigma, USA) and 300 IU/ml Deoxyribonuclease I (Sigma, USA), and then subjected to Percoll® (Sigma, USA) density gradient centrifugation to isolate villous CTB. In order to assure the purity of the cells, flow cytometry analysis was performed by using the trophoblast-specific epithelial cell marker cytokeratin 772 (link) (anti-cytokeratin 7, Dako, Switzerland). Vimentin (anti-vimentin, Sigma, USA), which is only expressed in potentially contaminating cells (e.g. mesenchymal cells, fibroblasts, smooth muscle cells, stromal cells)73 (link) served as a negative marker.
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