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Dnase 1 type 2

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DNase I type II is a lab equipment product. It is an enzyme that catalyzes the hydrolytic cleavage of DNA.

Automatically generated - may contain errors

Lab products found in correlation

Hyaluronidase type 5, by Merck Group (2 mentions) Dnase 1 type 2, by Roche (2 mentions) Liberase tm, by Roche (2 mentions) Collagenase type 4, by Worthington (2 mentions) Dynabead, by Thermo Fisher Scientific (2 mentions) Collagenase type xi, by Merck Group (1 mentions) Cacl2, by Fujifilm (1 mentions) Bsa fraction 5, by Fujifilm (1 mentions) Software, by FlowJo (1 mentions) Facsaria, by BD (1 mentions) Collagenase type 1, by Worthington (1 mentions) Zombie aqua live dead dye, by BioLegend (1 mentions) Fitc rat anti mouse cd45, by BioLegend (1 mentions) Facscanto b, by BD (1 mentions) Pe rat anti mouse cd4, by BioLegend (1 mentions) Apc rat anti mouse cd3, by BioLegend (1 mentions) Collagenase 4, by Worthington (1 mentions) Rnase type iiia, by Merck Group (1 mentions) Collagenase 2, by Merck Group (1 mentions) Facs calibur 2, by BD (1 mentions)

8 protocols using dnase 1 type 2

1

Single-Cell Analysis of Mouse Aortic Cells

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Single-cell suspensions were obtained from mouse abdominal aorta by enzyme dissociation.18 (link) Abdominal aorta between the diaphragm and bifurcation was freed of connective fat tissues. Aorta with adventitia was cut into 1-mm square pieces and treated for 60 minutes with the following enzyme solution: 125 U/mL of collagenase type XI (Sigma), 60 U/mL of type 1-s hyaluronidase (Sigma), 60 U/mL of type II DNase I (Sigma), 450 U/mL of type I collagenase (Worthington, Lakewood, NJ), 100 μmol/L CaCl2, and 0.1% BSA fraction V (Wako Pure Chemical Industries, Osaka, Japan) in PBS. The resulting single-cell suspension was treated with anti-CD16/32 and labeled with fluorescent dye-conjugated antibodies: BV421-CD45.2 (clone; 104), BV510-CD11b (clone; M1/70), PE-Ly6C (clone; HK1.4), PE-Cy7-F4/80 (clone; BM8), and APC-Cy7-Ly6G (clone; 1A8). Dead cells were labeled with 7AAD (7-amino-actinomycin D). All antibodies used in this assay were purchased from BioLegend (San Diego, CA). Flow cytometric analysis and sorting were performed using a FACSAria (BD Biosciences), and data were analyzed using FlowJo software (Flowjo, LLC, Ashland, OR).
Hiromi T., Yokoyama U., Kurotaki D., Mamun A., Ishiwata R., Ichikawa Y., Nishihara H., Umemura M., Fujita T., Yasuda S., Minami T., Goda M., Uchida K., Suzuki S., Takeuchi I., Masuda M., Breyer R.M., Tamura T, & Ishikawa Y. (2020). Excessive EP4 Signaling in Smooth Muscle Cells Induces Abdominal Aortic Aneurysm by Amplifying Inflammation. Arteriosclerosis, Thrombosis, and Vascular Biology, 40(6), 1559-1573.
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2

Tumor Immune Cell Profiling

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Tumors were harvested and digested with 20 ¼g/ml type II DNase I (Sigma, D4527) and 1 mg/mL collagenase IV (Worthington LS004186) to obtain single cell suspensions. Spleen and blood samples were processed with red blood cell lysis buffer (1 mM ammonium bicarbonate and 114 mM ammonium chloride). Antibodies (1:100 ratio) and Zombie Aqua™ live/dead dye (1:500 ratio) (Biolegend, 423102) were added to cell suspensions in PBS and incubated for 20 min on ice before flow analysis on BD FACS CantoB. Data were analyzed on FlowJo and represented as percentages of positive cells. Antibodies: FITC rat anti-mouse CD45 (Biolegend, 103108), APC rat anti-mouse CD3 (Biolegend, 100235), PE rat anti-mouse CD4 (Biolegend, 116005), and PE/Cyanine7 rat anti-mouse CD8a (Biolegend, 100721).
Xiao L., Somers K., Murray J., Pandher R., Karsa M., Ronca E., Bongers A., Terry R., Ehteda A., Gamble L.D., Issaeva N., Leonova K.I., O’Connor A., Mayoh C., Venkat P., Quek H., Brand J., Kusuma F.K., Pettitt J.A., Mosmann E., Kearns A., Eden G., Alfred S., Allan S., Zhai L., Kamili A., Gifford A.J., Carter D.R., Henderson M.J., Fletcher J.I., Marshall G., Johnstone R.W., Cesare A.J., Ziegler D.S., Gudkov A.V., Gurova K.V., Norris M.D, & Haber M. (2021). Dual targeting of chromatin stability by the curaxin CBL0137 and histone deacetylase inhibitor panobinostat shows significant preclinical efficacy in neuroblastoma. Clinical cancer research : an official journal of the American Association for Cancer Research, 27(15), 4338-4352.
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3

Optimal Tissue Digestion and Lymphocyte Enrichment

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First, tumors were finely minced and enzymatically digested for 25 min at 37°C in RPMI containing 1 mg/mL Collagenase type 4 (Worthington), 30 units/mL DNase I type II (Sigma-Aldrich) and 100 μg/mL hyaluronidase type V (Sigma-Aldrich). Lymph nodes and spleens were finely minced and digested for 10 min at 37°C with 2 U/mL Liberase TM (Roche) containing 30 units/mL DNase I type II. Subsequently, cell suspensions were passed through a 70 μm cell strainer and washed once with RPMI supplemented with 10% FCS, 1% penicillin, 1% streptomycin and 1% glutamax. Spleen digestions were subjected to ACK lysis and, together with the lymph node digestions, washed twice before use in subsequent experiments. To enrich for the lymphocytes and to remove dead cell debris from tumor digestions, the tumor digestion mixture was loaded on a Ficoll gradient. The interface was collected, washed twice and used for subsequent flow cytometric analysis.
Cornelissen L.A., Blanas A., Zaal A., van der Horst J.C., Kruijssen L.J., O’Toole T., van Kooyk Y, & van Vliet S.J. (2020). Tn Antigen Expression Contributes to an Immune Suppressive Microenvironment and Drives Tumor Growth in Colorectal Cancer. Frontiers in Oncology, 10, 1622.
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4

Retinal Vascular Isolation Protocol

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Retinas were incubated with 1 mL of deionized water and gently rotated for 60 min at room temperature. Then, 200 U DNase I Type II (Sigma-Aldrich, USA) was used to dissociate the lysed cell debris from retinal vessels for 10 min at 37°C. Retinal vessels were then incubated with the Dynabeads (Invitrogen, USA) conjugated with mouse anti-PECAM-1 antibody (BD Bioscience, USA) for 30 min at 4°C, followed by incubating with the sheep anti-mouse IgG-conjugated magnetic beads (Dynabead; Invitrogen, USA). Retinal vessels were obtained via magnetic separation and lysed for RNA extraction [15 (link)].
Ma C., Shi Z.H., Han X.Y., Liu C., Yan B, & Du J.L. (2022). Targeting circRNA-MAP4K2 for the treatment of diabetes-induced retinal vascular dysfunction. Aging (Albany NY), 14(15), 6255-6268.
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5

Isolated Extracellular Matrix Extraction

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ECM isolation was modified from published protocols for adipose and lung tissues (8–10 (link)). VAT explants (1 g) were freeze-thawed in 10 mM Tris, 5 mM EDTA, and 1% phenylmethanesulphonylfluoride (PMSF) (pH 8.0) from −80°C, 20 minutes to 37°C, 5 minutes 3 times. Explants were incubated at 37°C for 24 hours in 0.25% Trypsin/0.1% EDTA; washed in rinsing buffer (8 g/L NaCl, 200 mg/L KCl, 1 g/L Na2HPO4, 200 mg/L KH2PO4, 1% PMSF) at 37°C for 20 minutes three times; incubated at 37°C for 24 hours in 55 mM Na2HPO4, 17 mM KH2PO4, 4.9 mM MgSO4⋅7H2O, 160 U/mL DNase I type II, 100 μg/mL RNase type IIIA, 80 U/mL lipase type VI-S (Sigma-Aldrich, Inc., St. Louis MO, USA), and 1% PMSF; and washed sequentially in rinsing buffer at 37°C for 20 minutes three times; once in 99.9% isopropanol, 1% PMSF at 25°C for 24 hours; rinsing buffer at 37°C for 20 minutes three times; 70% EtOH at 37°C for 20 minutes three times; and storage solution (PBS, 1% PMSF) at 37°C for 20 minutes once. Explants were stored in storage solution 4°C.
Baker N.A., Muir L.A., Washabaugh A.R., Neeley C.K., Chen S.Y., Flesher C.G., Vorwald J., Finks J.F., Ghaferi A.A., Mulholland M.W., Varban O.A., Lumeng C.N, & O’Rourke R.W. (2017). Diabetes-Specific Regulation of Adipocyte Metabolism by the Adipose Tissue Extracellular Matrix. The Journal of Clinical Endocrinology and Metabolism, 102(3), 1032-1043.
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6

Tumor and Lymph Node Dissociation

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Tumors were finely minced and enzymatically digested in RPMI containing 1 mg/mL Collagenase type 4 (Worthington), 30 units/mL DNase I type II (Sigma‐Aldrich) and 100 μg/mL hyaluronidase type V (Sigma‐Aldrich) for 25 min at 37 °C. Lymph nodes were digested with 2 U/mL Liberase TM (Roche) enriched with 30 units/mL DNase I type II for 10 min at 37 °C. All cell suspensions were passed through a 70 μm cell strainer and washed with RPMI supplemented with 10% FCS, 1% penicillin, 1% streptomycin and 1% glutamax. Lymph node digestions were washed twice and directly used for subsequent experiments. Tumor digestion mixtures were loaded on a Ficoll gradient to enrich for lymphocytes and to remove dead cell debris.
Cornelissen L.A., Blanas A., van der Horst J.C., Kruijssen L., Zaal A., O'Toole T., Wiercx L., van Kooyk Y, & van Vliet S.J. (2019). Disruption of sialic acid metabolism drives tumor growth by augmenting CD8+ T cell apoptosis. International Journal of Cancer, 144(9), 2290-2302.
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7

Colon Cancer Single-Cell Isolation

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Colon CSC was prepared by generating a single-cell suspension from human colon cancer tissues. Tumor tissues were finely cut and minced with scalpel in 5 ml of stem cell medium. The tissue suspension was further disaggregated by vigorous pipetting. Enzymatic disaggregation was achieved by incubating the tissues for 2 hours at 37°C with collagenase II (1.5 mg/ml) and DNase I type II (600 U/ml)(Sigma Aldrich, France). Passage through a Ficoll column removed necrotic cells, debris, and red blood cells. The population of mononuclear cells obtained was then resuspended in 5 ml of stem cell medium and serially filtered by using sterile gauze 100-μm, 70-μm, and 40-μm nylon meshes.
Becherirat S., Valamanesh F., Karimi M., Faussat A.M., Launay J.M., Pimpie C., Therwath A, & Pocard M. (2018). Discontinuous Schedule of Bevacizumab in Colorectal Cancer Induces Accelerated Tumor Growth and Phenotypic Changes. Translational Oncology, 11(2), 406-415.
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8

Tumor-Infiltrating Lymphocyte Isolation Protocol

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Tumor-infiltrating lymphocytes were obtained by incubating excised tumor tissues for 30 min at 37°C with 1 μg/ml Liberase TL, research grade (Roche) and 50 μg/ml DNase I, type II (Sigma). Cells were subjected to flow cytometry on a BD FACSCanto II or BD FACSCalibur II, and the resultant data were analyzed with the FlowJo software.
Gong Y., Suzuki T., Kozono H., Kubo M, & Nakano N. (2020). Tumor-infiltrating CD62L+PD-1-CD8 T cells retain proliferative potential via Bcl6 expression and replenish effector T cells within the tumor. PLoS ONE, 15(8), e0237646.
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