Deoxyribonuclease 1

Manufactured by AppliChem

Sourced in Germany

Deoxyribonuclease I (DNase I) is an enzyme that catalyzes the hydrolytic cleavage of DNA. It acts by cleaving the phosphodiester bonds within the DNA molecule, resulting in the formation of shorter DNA fragments.

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Lab products found in correlation

Aminoguanidine hydrochloride, by Merck Group (1 mentions) Gentlemacs c tube, by Miltenyi Biotec (1 mentions) Percoll gradient, by Thermo Fisher Scientific (1 mentions) 2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions) Penicillin streptomycin glutamine (psg), by Thermo Fisher Scientific (1 mentions) Macs smartstrainer, by Miltenyi Biotec (1 mentions) Gentlemacs dissociator, by Miltenyi Biotec (1 mentions) Liberase, by Roche (1 mentions) Pronase e, by Merck Group (1 mentions) Collagenase 2, by Worthington (1 mentions) Lsrfortessa x 20 flow cytometer, by BD (1 mentions) Dispase 2, by Merck Group (1 mentions) 7 aad viability staining solution, by Thermo Fisher Scientific (1 mentions) Hank s balanced salt solution, by Merck Group (1 mentions) Papain, by Merck Group (1 mentions) Mgso4, by Merck Group (1 mentions) Cytometric bead array, by BD (1 mentions) Collagenase d, by Merck Group (1 mentions) Dispase, by Roche (1 mentions) Hepes, by Lonza (1 mentions)

4 protocols using deoxyribonuclease 1

Isolation and Processing of Lung and Liver Lymphocytes

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Lungs were harvested and diced into gentleMACS C Tubes (Miltenyi Biotec) and washed down with 3 ml of media [RPMI, 10 mM aminoguanidine hydrochloride (Sigma-Aldrich), 10 mM Hepes, penicillin-streptomycin-glutamine (100×, Gibco), 2-mercaptoethanol (50 μM, Gibco)] devoid of fetal calf serum (FCS) and EDTA prewarmed in a 37°C water bath. Digestion mix containing liberase (33.3 μg/ml; Roche, #05401020001) and deoxyribonuclease I (68 μg/ml; Applichem) was added. Lungs were dissociated on a gentleMACS Dissociator (Miltenyi Biotec) and placed in a shaking incubator at 37°C for 30 min. Lungs were dissociated again and mashed through 70-μm MACS SmartStrainers (Miltenyi Biotec). Red blood cells were lysed with 139.5 mM NH₄Cl and 17 mM tris-HCl (Erylysis buffer). For further processing, media containing 2% FCS and 1 mM EDTA were used. Mouse mediastinal LNs were harvested and mashed through a 100-μm Corning nylon cell strainer.
For LCMV-infected liver, perfusion with PBS was done till blood was removed. Organs were harvested and mashed through a 100-μm cell strainer in RPMI 1640 containing 10% FCS. Lymphocytes were purified on a 70%/40% Percoll gradient (Fisher Scientific). Purified lymphocytes were further processed for scRNA-seq as described previously (22 (link)).

Swarnalekha N., Schreiner D., Litzler L.C., Iftikhar S., Kirchmeier D., künzli M., Son Y.M., Sun J., Moreira E.A, & King C.G. (2021). T resident helper cells promote humoral responses in the lung. Science immunology, 6(55), eabb6808.

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Isolation of Podocytes from Transgenic Mice

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Glomerular preparation and isolation of GFP-positive (GFP⁺) podocytes from 6- to 8-week-old mT/mG-Nphs2cre and WT1^flox/flox/icre/Tdtomato mice were done, as described previously (4 (link)). Renal arteries were perfused with Dynabeads M-450 in Hank’s balanced salt solution (HBSS), and dissected kidneys were minced and incubated in digestion solution for 15 min at 37°C [collagenase II (300 U/ml; Worthington), pronase E (1 mg/ml; Sigma-Aldrich), and deoxyribonuclease I (50 U/μl; AppliChem) in HBSS]. The digest was passed through 100-μm sieves twice, washed with HBSS, and spun down, and glomeruli were isolated using a magnetic concentrator. Glomeruli were dissociated into a single-cell suspension by incubation in digestion solution at 37°C on an incubator shaker for 40 min. Cells were sieved through a 40-μm filter, and GFP⁺ cells were FACS sorted on a FACS MoFlo flow cytometer.

Ettou S., Jung Y.L., Miyoshi T., Jain D., Hiratsuka K., Schumacher V., Taglienti M.E., Morizane R., Park P.J, & Kreidberg J.A. (2020). Epigenetic transcriptional reprogramming by WT1 mediates a repair response during podocyte injury. Science Advances, 6(30), eabb5460.

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Zebrafish Embryo Dissociation and Flow Cytometry

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Pooled zebrafish embryos from each treatment group were killed on ice then transferred to calcium- and magnesium-free Hank’s balanced salt solution (Sigma-Aldrich). After an initial mechanical dissociation (trituration through a yellow micropipette tip), embryo fragments were pelleted at 1500 rpm before being resuspended in 0.01% papain (Sigma-Aldrich), 0.1% dispase II (Sigma-Aldrich), 0.01% deoxyribonuclease I (AppliChem GmbH, Darmstadt, Germany) and 12.4mM MgSO₄ (Sigma-Aldrich) in calcium- and magnesium-free Hank’s balanced salt solution for 15 min at room temperature with trituration through a micropipette tip every 5 min. Dissociated cells were centrifugated through a 20 µm fine mesh filter then resuspended in 140 mM NaCl, 3.33 mM CaCl₂ and 10 mM Hepes. Immunostaining for flow cytometry was performed in parallel on all cell samples with Alexa Fluor^® 488 anti-human CD19 antibody (BioLegend, San Diego, CA, USA) for graft cell identity, and 7-AAD viability staining solution (eBioscience™ from Thermo Fischer Scientific) and fluorescein isothiocyanate-conjugated annexin A5 (BioLegend) to assess viability. The identity of single cells was determined by the fluorescence intensity for the 3 markers on a Becton Dickinson LSRFortessa™ X-20 flow cytometer and results were analyzed with FlowJo software (Becton Dickinson, Heidelberg, Germany).

Gauert A., Olk N., Pimentel-Gutiérrez H., Astrahantseff K., Jensen L.D., Cao Y., Eggert A., Eckert C, & Hagemann A.I. (2020). Fast, In Vivo Model for Drug-Response Prediction in Patients with B-Cell Precursor Acute Lymphoblastic Leukemia. Cancers, 12(7), 1883.

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Omentum and Mesentery Fibroblast Isolation

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Omentum or mesentery was obtained and transferred into a 24-well dish filled with RPMI 1640 medium containing 2% fetal calf serum (FCS), 20 mM Hepes (all from Lonza), collagenase D (1 mg/ml; Sigma-Aldrich), deoxyribonuclease I (25 g/ml; Applichem), and Dispase (Roche). Dissociated tissues were incubated at 37°C for 30 min. After enzymatic digestion, cell suspensions were washed with phosphatebuffered saline (PBS) containing 0.5% FCS and 10 mM EDTA. For in vitro assays, omental or mesenteric fibroblastic stromal cells were cultured for 7 days in RPMI 1640/10% FCS and 5 × 10 4 cells were stimulated with lipopolysaccharide (LPS; 1 g/ml), OmpC/F (10 g/ml), or Pam3CSK (15 g/ml) or left untreated (medium). Supernatants were collected after 24 hours, and TNF and CCL2 concentrations were determined using cytometric bead array (BD Biosciences).

Perez-Shibayama C., Gil-Cruz C., Cheng H.W., Onder L., Printz A., Mörbe U., Novkovic M., Li C., Lopez-Macias C., Buechler M.B., Turley S.J., Mack M., Soneson C., Robinson M.D., Scandella E., Gommerman J, & Ludewig B. (2018). Fibroblastic reticular cells initiate immune responses in visceral adipose tissues and secure peritoneal immunity. Science immunology, 3(26).

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