Dnase type 1

Manufactured by Roche

Sourced in United States, Switzerland, France

DNase (Type 1) is a laboratory reagent used to degrade DNA molecules. It catalyzes the hydrolytic cleavage of phosphodiester linkages in the DNA backbone, effectively breaking down DNA strands.

Automatically generated - may contain errors

Lab products found in correlation

Collagenase type d, by Roche (7 mentions) 70 μm sieve, by BD (6 mentions) Collagenase type 4, by Roche (5 mentions) Anti cd16 32 clone 93, by BioLegend (2 mentions) Rbc lysis buffer, by Thermo Fisher Scientific (2 mentions) Flavoperidol, by Merck Group (2 mentions) Zombie aqua viability dye, by BioLegend (2 mentions) Facsaria 2 flow cytometer, by BD (2 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Collagenase type 3, by Worthington (2 mentions) True nuclear transcription factor buffer set, by BioLegend (2 mentions) Pe labeled rat anti mouse cd8a antibody, by BioLegend (2 mentions) Apc labeled anti mouse cd45 antibody, by BioLegend (2 mentions) Collagenase type 1, by Thermo Fisher Scientific (2 mentions) Nuclease free water, by Thermo Fisher Scientific (2 mentions) Nanodrop 2000 uv vis spectrophotometer, by Thermo Fisher Scientific (2 mentions) Tri reagent, by Merck Group (2 mentions) Collagenase type 4, by Merck Group (2 mentions) Collagenase a, by Roche (2 mentions)

Close spelling variants of this product

DNase I type II DNase I Type IV DNase I DNase

29 protocols using dnase type 1

Isolation of Lung and Blood Cells

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The airways were washed three times with 400μl of PBS and
bronchoalveolar lavage fluid was collected. Following centrifugation of the BAL
fluid supernatants were stored at -80°C for further analysis and cells
were resuspended in 500μl of complete RPMI media (10% FCS, 2mM
L-glutamine, 100U/ml penicillin/streptomycin) (GIBCO, Life Technologies). For
lung cell isolation, the left lung lobes were cut into small pieces and digested
in complete media supplemented with 0.15mg/ml collagenase (Type D; Roche
Diagnostics) and 25μg/ml DNase (Type 1; Roche Diagnostics) for 1h at
37°C. Lung homogenate obtained was then filtered through a 70μm
sieve (BD Bioscience), washed, and resuspended in 1ml of complete media.
Erylysis was performed on 200μl whole blood and leukocytes were washed
twice and then then resuspended in 1ml of complete media.

Puttur F., Denney L., Gregory L.G., Vuononvirta J., Oliver R., Entwistle L.J., Walker S.A., Headley M.B., McGhee E.J., Pease J.E., Krummel M.F., Carlin L.M, & Lloyd C.M. (2019). Pulmonary environmental cues drive group 2 innate lymphoid cell dynamics in mice and humans. Science immunology, 4(36), eaav7638.

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Isolation and Characterization of Lung Mononuclear Phagocytes

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Mice were euthanized with carbon dioxide. Lungs were perfused with PBS trough the right ventricle until no blood was visible in the tissue. Lungs were removed, minced and placed in a digestion buffer containing collagenase type 4 (1.6 mg/ml; Roche), DNase type 1 (50 units/ml; Roche) and Flavoperidol (1 μM, Sigma-Aldrich) in RPMI 1640 medium. Lungs were incubated at 37 °C for 15 min while on a rotator. Red blood cells were lysed using RBC lysis buffer (eBioscience) at 4 °C for a 5 min incubation. The obtained cell suspension was filtered using a 70-micron filter, washed and cells were resuspended in PBS. Cells were then incubated with anti-Fc blocking mAb (anti CD16/32, clone 93, BioLegend) and viability dye Zombie Aqua (BioLegend) for 10 min. After this, cells where stained with an antibody cocktail containing APC Cy7-conjugated CD45, PE-conjugated F4/80, BV605-conjugated CD11c, v450-conjugated CD11b, PE Cy7-conjugated CD64, AF700-conjufgated Ly6c, FITC-conjugated Ly6g, AF647-conjugated SiglecF mAb for 30 min. All labeled cells were passed through a FACS Aria II flow cytometer (BD Biosciences) and after proper gating 4 subsets of lung MPs were sorted^{29 (link)}. Data were analyzed with FlowJo 8.3.3 software (TreeStar Inc.). The antibodies used for FACS are listed in Supplementary Table 1.

Sajti E., Link V.M., Ouyang Z., Spann N.J., Westin E., Romanoski C.E., Fonseca G.J., Prince L.S, & Glass C.K. (2020). Transcriptomic and epigenetic mechanisms underlying myeloid diversity in the lung. Nature immunology, 21(2), 221-231.

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Airway and Lung Tissue Sampling

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Airway Lumen Bronchoalveolar lavage (BAL) was performed by three flushings of the lung with 0.4 mL PBS via a tracheal cannula resulting in the recovery of 1 mL BAL fluid. Lung parenchyma one lobe of lung tissue was digested in complete media (RPMI + 10% FCS, 2 mm L-Glutamine, 100 U/mL Penicillin/Streptomycin) containing 0.15 mg/mL collagenase (Type D, Roche Diagnostics) and 25 μg/mL DNase (Type 1, Roche Diagnostics). Cells were recovered by filtration through a 70-μm nylon sieve (Falcon) and resuspended in 1-mL complete media.

Murdoch J.R., Gregory L.G, & Lloyd C.M. (2014). γδT cells regulate chronic airway inflammation and development of airway remodelling. Clinical and Experimental Allergy, 44(11), 1386-1398.

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Isolation and Analysis of Lung Immune Cells

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After the removal of the left lobe of mice lung, the tissue was chopped and digested with 15 mg/mL collagenase (Type D; Roche Diagnostics) and 25 μg/mL DNase (Type 1; Roche Diagnostics) in 4 mL RPMI + 10% CFS for 1 h at 37 °C with agitation. Lung was then passed through a 70 μm sieve (BD Bioscience). The resulting cell suspension was washed, red blood cells were lysed, and the remaining cells were resuspended in 1 mL RPMI. To analyze cell populations, the cell suspension was labeled using monoclonal antibodies anti-CD45 (clone 30-F11) (1:100) anti-Ly6G (clone 1A8) (1:400), anti-F4/80 (clone BM8) (1:100), anti-CCR5 (HM-CCR5 (7A4)) (1:200) from eBioscience for 30 min. All data was acquired by flow cytometry (FACSCalibur; BD Biosciences PharMingen) and analyzed using FlowJo software (Tree Star, Inc., Ashland, OR, USA). Gating strategies for the identification of neutrophils and macrophages in these experiments are shown in Figure S2.

Ferrero M.R., Garcia C.C., Dutra de Almeida M., Torres Braz da Silva J., Bianchi Reis Insuela D., Teixeira Ferreira T.P., de Sá Coutinho D., Trindade de Azevedo C., Machado Rodrigues e Silva P, & Martins M.A. (2021). CCR5 Antagonist Maraviroc Inhibits Acute Exacerbation of Lung Inflammation Triggered by Influenza Virus in Cigarette Smoke-Exposed Mice. Pharmaceuticals, 14(7), 620.

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Isolation and Characterization of Lung Mononuclear Phagocytes

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Detailed Liver Cell Isolation Protocol

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For liver digestion, we used Collagenase type 4 (270 units/mg and 290 units/mg) (Worthington Biochemical Corporation, Lakewood, CA, USA), DNase type 1 (2000 units/mg) (Roche Diagnostics, Risch, Switzerland), Heparin (Drossapharm, Arlesheim, Switzerland), and 40% glucose (Grosse Apotheke Dr. G. Bichsel AG, Interlaken, Switzerland). Culture grade powders for Grey’s Balanced Salt Solution (GBSS) with or without NaCl were from Sigma-Aldrich (Buchs, Switzerland). Nycodenz density gradient solution was from Axon lab AG (Le Mont-sur-Lausanne, Switzerland). William’s E medium with Glutamax, Calcium-free Dulbecco’s phosphate buffered saline (DPBS), Hank’s hank balanced salt solution without calcium, Iscove’s Modified Dulbecco’s Medium (IMDM) with Glutamax supplement, HEPES buffer solution, fetal bovine serum (FBS), penicillin-streptomycin, trypsin, and EDTA were purchased from Life Technologies (Grand Island, USA). Oil-Red-O solution was obtained from Sigma-Aldrich. Fixation buffer was purchased from Biolegend (San Diego, CA, USA). Perm/Wash was obtained from Becton Dickinson. Paraformaldehyde solution for cell fixation was acquired from Sigma-Aldrich. Custom wash solution, STOP solution, and MACS buffer were identical to our previous report [25 (link)]. Recombinant human TGF-β1 was obtained from PreproTech (Rocky Hill, CT, USA).

Balaphas A., Meyer J., Gameiro C., Frobert A., Giraud M.N., Egger B., Bühler L.H, & Gonelle-Gispert C. (2022). Optimized Isolation and Characterization of C57BL/6 Mouse Hepatic Stellate Cells. Cells, 11(9), 1379.

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Isolation of respiratory cells

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BAL was obtained by washing the airways three times with a volume of 400μl PBS. After centrifugation the cell pellet was resuspended in 500μl of complete media (RPMI, 10%FCS, 2mM L-glutamine, 100U/ml penicillin/streptomycin) (GIBCO, Life Technologies) and the supernatant stored at -80°C for further analysis. The left lobe was finely chopped and digested with 15 mg/ml collagenase (Type D; Roche Diagnostics) and 25μg/ml DNase (Type 1; Roche Diagnostics) in 4ml complete media for 1hr at 37°C with agitation. Lung was then passed through a 70μm sieve (BD Bioscience), cells were washed, red blood cells lysed and the cell pellet resuspended in 1ml complete media. Airway epithelial cells were obtained by tracheal brushing using 4mm interdental brushes (TePe).

Denney L., Branchett W., Gregory L.G., Oliver R.A, & Lloyd C.M. (2017). Epithelial derived TGF-β1 acts as a pro-viral factor in the lung during influenza A infection. Mucosal immunology, 11(2), 523-535.

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Isolation of Lung Immune Cells

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Bronchoalveolar lavage (BAL) was performed with PBS via a tracheal
cannula. The volume of BAL fluid instilled was 3 x 200μl aliquots for
neonatal mice, 3 x 300 μl for 3 week old mice and 3 x 400μl in adults (29 (link)). After lavage, the large left lobe
of the lung was mechanically chopped and incubated at 37°C for 1 hour in
complete media (RPMI + 10% fetal calf serum, 2mM L-glutamine, 100U/ml
penicillin/streptomycin) containing 0.15mg/mL collagenase (Type D, Roche
Diagnostics) and 25mg/mL DNAse (Type 1, Roche Diagnostics). Cells were
recovered by filtration through a 70-μm nylon sieve (Falcon, BD Biosciences,
MA), washed twice, resuspended in 1ml complete media, and counted in a
haemocytometer (Immune Systems). The total cell yield was quantified by
haemocytometer. All cell counts were performed blind by the same
observer.

Saglani S., Gregory L.G., Manghera A.K., Branchett W.J., Uwadiae F., Entwhistle L.J., Oliver R.A., Vasiliou J.E., Sherburn R., Lui S., Puttur F., D V., Walker S.A., Buckley J., Grychtol R., Fainardi V., Denney L., Byrne A., von Mutius E., Bush A, & Lloyd C.M. (2018). Inception of early life allergen induced airway hyperresponsiveness is reliant on IL-13+CD4+ T cells. Science immunology, 3(27), eaan4128.

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Isolation of Lung Cells from Bronchoalveolar Lavage

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Bronchoalveolar lavage (BAL) was performed using three aliquots of saline via a tracheal cannula (28 (link)). BAL fluid was centrifuged, supernatants removed, and cell pellets resuspended in 0.5 ml complete media (RPMI/10% FCS/2 mM l-glutamine/100 U/ml penicillin/streptomycin). One lobe of lung tissue was mechanically chopped and incubated at 37°C for 1 h in complete media containing 0.15 mg/ml collagenase (Type D; Roche Diagnostics, West Sussex, UK) and 25 μg/ml DNAse (Type 1; Roche Diagnostics). Recovered cells were filtered through a 70-um nylon sieve, washed twice and resuspended in 1 ml complete media.

Vasiliou J.E., Lui S., Walker S.A., Chohan V., Xystrakis E., Bush A., Hawrylowicz C.M., Saglani S, & Lloyd C.M. (2014). Vitamin D deficiency induces Th2 skewing and eosinophilia in neonatal allergic airways disease. Allergy, 69(10), 1380-1389.

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Lung Cell Isolation Protocol

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Freshly excised lungs were kept in cold FACS -fluorescence-activated cell sorting- buffer (Phosphate-buffered saline accompanied with 0.2% Bovine serum albumin, 0.02% Sodium azide and 5 mM EDTA). With surgical scissors, the main airways were removed and the lungs were cut into small portions before enzymatic digestion, for 45 min, in 1 mL of dissociation media consisting of HBSS- Hanks’ Balanced Salt Solution- (Sigma-Aldrich, St. Louis, USA) containing 3 mg/mL collagenase/dispase (Roche Applied Science, Penzberg, Germany) and 0.2 mg/mL DNase type 1 (Roche Applied Science). To eliminate red blood cells, the lung single cell suspensions were incubated in 1 mL of red blood cell lysis buffer (8.3 g/L NH4Cl, 10 mM Tris-HCl, and pH 7.5) for 1 min. Samples were filtered through a 100 μm nylon cell strainer (BD Bioscience, San Jose, USA) before markers labelling.

Al-Rubaie A., Wise A.F., Sozo F., De Matteo R., Samuel C.S., Harding R, & Ricardo S.D. (2018). The therapeutic effect of mesenchymal stem cells on pulmonary myeloid cells following neonatal hyperoxic lung injury in mice. Respiratory Research, 19, 114.

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