According to the previous experimental method 22) , briefly, Ti substrates were immersed in dopamine (4 mg/mL, H8502-5G, Sigma-Aldrich, San Francisco, CA, USA) for 24 h at room temperature to obtain the polydopaminecoated samples (Ti-PDOP). Then Ti-PDOP substrates were immersed in DNase I (25 units/mL, Sigma-Aldrich) or the inactivated DNase I solution for 6 h at room temperature to obtained the DNase I-polydopaminecoated samples and inactivated DNase I-polydopaminecoated samples, denoted by Ti-PDOP-DNase I and Ti-PDOP-inactivated DNase I, respectively.
Dnase 1
Manufactured by Merck Group
Sourced in United States, Germany, Switzerland, United Kingdom, Italy, Japan, Macao, Canada, Sao Tome and Principe, China, France, Australia, Spain, Belgium, Netherlands, Israel, Sweden, India
DNase I is a laboratory enzyme that functions 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 4, by Merck Group (260 mentions)
Collagenase d, by Roche (232 mentions)
Hyaluronidase, by Merck Group (189 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (188 mentions)
Collagenase d, by Merck Group (157 mentions)
Liberase, by Roche (149 mentions)
Percoll, by GE Healthcare (138 mentions)
Collagenase type 4, by Merck Group (127 mentions)
Collagenase, by Merck Group (121 mentions)
Trizol reagent, by Thermo Fisher Scientific (108 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (99 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (94 mentions)
Collagenase 1, by Merck Group (94 mentions)
Collagenase 4, by Thermo Fisher Scientific (93 mentions)
Liberase tl, by Roche (87 mentions)
Trypsin, by Thermo Fisher Scientific (85 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (84 mentions)
Collagenase type 4, by Worthington (75 mentions)
Collagenase a, by Roche (75 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (72 mentions)
4 163 protocols using dnase 1
1
Polydopamine-Coated Titanium Substrates
2
Isolation and Characterization of Immune Cells
3
DNase I Digestion Assay Protocol
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DNase I digestion assay was described previously (Ye et al., 2017 (link)). In brief, nuclei were purified from α4β7+ progenitors (Lin−CD127+c-KitintSca-1intα4β7+) according to the manufacturer’s protocol with a nuclei isolating kit (Sigma-Aldrich). Then, nuclei were resuspended with DNase I digestion buffer and treated with indicated units of DNase I (Sigma-Aldrich) at 37°C for 5 min 2 × DNase I stop buffer (20 mM Tris, pH 8.0, 4 mM EDTA, and 2 mM EGTA) was added to stop reactions. DNA was extracted and examined by qPCR.
4
Isolation of Murine Sertoli Cells
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A modified previously described method was used to isolate Sertoli cells from the testes of 21-dpp mice22 (link)23 (link). Briefly, testes were decapsulated under a dissection microscope. The seminiferous tubules were pooled and washed with phosphate-buffered saline (PBS) thrice and incubated with 2 mg/ml collagenase I (Sigma, C7661, MO, USA) and 0.5 mg/ml DNase I (Sigma, D4527) in DMEM: F12 (HyClone, SH40007-13, UT, USA) for 15 minutes at 37°C on a shaker, then washed twice with DMEM: F12 and further digested with 2 mg/ml collagenase I, 0.5 mg/ml DNase I and 1 mg/ml hyaluronidase (Sigma, H3506) for 15 minutes at 37°C. The tubules were allowed to settle and were then washed twice with DMEM: F12 before being digested with 2 mg/ml collagenase I, 0.5 mg/ml DNase I, 2 mg/ml hyaluronidase and 1 mg/ml trypsin (Sigma, T8003) for 30 minutes at 37°C. These dispersed cells were then washed twice with DMEM: F12 medium and placed into culture dishes in DMEM: F12 containing 10% fetal bovine serum (HyClone, SV30087-02) and were incubated at 37°C with 5% CO2. After 1 day of culturing, the solution was removed, and the cells were treated with a hypotonic solution (20 mM Tris, pH 7.4) for 1 minutes for the removal of remaining germ cells, if any, and harvested for further analyses.
5
Investigating eDNA Role in Symplasmata
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To study the role of eDNA in YS19 symplasmata stability, the bacterium was cultivated in LB liquid medium for a certain time, then the cultures were treated with DNase I (10 mg mL -1 , Sigma Aldrich) and incubated at 37∞C for 1 h. For excluding other effects beside the enzymatic function of DNase I, the heat-inactivated DNase I (1 h at 75∞C) was used as a control. Then statistical analysis of the symplasmata formation ratio and average size were carried out. All the experiments were performed in triplicate.
6
Isolation and Characterization of Immune Cells
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Lymphoid and non-lymphoid organ DCs were harvested and prepared as described previously30 (link). Briefly, spleens and inguinal skin-draining LNs were minced and digested in 5 ml of IMDM +10% FCS (cIMDM) with 250 μg/ml of collagenase B (Roche) and 30 U/ml of DNaseI (Sigma-Aldrich) for 45 min at 37 °C with stirring. Lungs were minced and digested in 5 ml of cIMDM with 4 mg/ml of collagenase D (Roche) and 30 U/ml of DNaseI (Sigma-Aldrich) for 1.5 h at 37 °C with stirring. Tumours were minced and digested in serum-free IMDM with 125 μg/ml Liberase (Roche) and 30 U/mL of DNaseI (Sigma-Aldrich) for 45 minutes at 37 °C with stirring. After digestion was complete, single-cell suspensions from all organs were passed through 70-μm strainers and red blood cells were lysed with ammonium chloride–potassium bicarbonate (ACK) lysis buffer. Cells were subsequently counted with a Vi-CELL analyzer (Beckman Coulter) and 3–5×106 cells were used per antibody staining reaction.
7
Isolation of Rhesus Monkey Germ Cells
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Testis tissues from each rhesus monkey were minced and digested with Enzyme Solution I (20 ml of DMEM [Cat#11965‐084, Gibco], 80 mg of collagenase IV [Cat#C5138, Sigma], 20 mg of DNase I [Cat#DN25, Sigma], 50 mg of hyaluronidase [Cat#H3884, Sigma], and 200 μl of penicillin‐streptomycin [Cat#15140‐122, Gibco]) for 30 min at 37°C. Cells were centrifuged at 600 × g for 5 min at 4°C, and the supernatant was discarded. We then incubated the cells with Enzyme Solution II (20 ml of 0.25% trypsin‐EDTA [Cat#25200‐114, Gibco], 80 mg of collagenase IV [Cat#C5138, Sigma], 20 mg of DNase I [Cat#DN25, Sigma], 50 mg of hyaluronidase [Cat#H3884, Sigma], and 200 μl of penicillin‐streptomycin [Cat#15140‐122, Gibco]) for 15–20 min (5‐year‐old monkey) or 28 min (6‐M‐old monkeys) at 37°C. We filtered the cell suspension with a 100‐μm strainer following FBS addition, and 2 ml of red blood cell lysis buffer was added and the suspension incubated for 5 min at 4°C. After filtration through a 40‐μm strainer, germ cells were resuspended in 25 ml of wash buffer (50 ml of HBSS [Cat#14175‐079, Gibco], 50 mg of DNase I [Cat#DN25, Sigma], and 500 μl of penicillin‐streptomycin [Cat#15140‐122, Gibco]) for use in the STA‐PUT method.
8
Multimodal Immune Profiling in Mice
9
Immune Profiling of Tumor Samples
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For immune-profiling, tumors were cut into small pieces (1 mm3), treated with 1 mg/mL collagenase A (MilliporeSigma, Burlington, MA, USA) and 250 units/mL DNase I (MilliporeSigma) at 37°C for 30 min, and filtered through a 40 μm strainer. The homogenate was layered onto Lympholyte M (CEDARLANE, Burlington, NC, USA). Buffy coat was incubated with Zombie Aqua viability kit (Biolegend), Fc-block (Biolegend), followed by the primary antibodies. Following fixation with FluoroFix Buffer (Biolegend), the data were collected on the FACSCelesta instrument (BD Biosciences, San Jose, CA, USA), analyzed by the BD FACSDiva software (BD Biosciences) and the FlowJo software version 10 (BD Biosciences). To quantify mCherry expressing breast cancer cells, the lungs were digested with 62.5 μg/mL Liberase (MilliporeSigma) and 500 units/mL DNase I (MilliporeSigma). The cell suspension was incubated with Zombie Aqua viability kit (Biolegend) and analyzed using a S1000EON benchtop flow cytometer (Stratedigm, San Jose, CA, USA).
10
Tissue Digestion and Cell Isolation
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Mouse tissue -In some experiments mice challenged with CT26 or EG7 had their tumors excised and digested with 0.5 WU/mL Liberase DL (Roche) and 50 mg/mL DNaseI (Roche) for 20-45 min at 37 C. Cells were then passed through a 100 mm cell strainer and used for assays directly or tumor infiltrating lymphocytes were isolated using percoll gradients of 40% and 70%.
Human tissue -Ascitic fluid was assessed directly as a single cell suspension. Ovarian tumor samples were cut into small pieces and incubated in supplemented RPMI1640 with DNase I (Sigma) and Liberase TM (Roche Diagnostics) for 20 min at 37 C. Remaining tissue was mechanically dissociated and, together with the cell suspension, passed through a 70 mm cell strainer. Samples of freshly excised cutaneous squamous cell carcinoma (cSCC) and normal skin were minced and treated with 1mg/mL collagenase IA (Sigma) and 10 mg/mL DNase I (Sigma) in RPMI medium (GIBCO) at 37 C for 1.5 hours before straining through a 70 mm cell filter (BD) and centrifugation (600 x g, 20 min) over an Optiprep (Axis-Shield) density gradient. Matched peripheral blood samples were obtained and peripheral blood mononuclear cells were separated by centrifugation over Lymphoprep (Axis-Shield) at 600 x g for 30 min.
Human tissue -Ascitic fluid was assessed directly as a single cell suspension. Ovarian tumor samples were cut into small pieces and incubated in supplemented RPMI1640 with DNase I (Sigma) and Liberase TM (Roche Diagnostics) for 20 min at 37 C. Remaining tissue was mechanically dissociated and, together with the cell suspension, passed through a 70 mm cell strainer. Samples of freshly excised cutaneous squamous cell carcinoma (cSCC) and normal skin were minced and treated with 1mg/mL collagenase IA (Sigma) and 10 mg/mL DNase I (Sigma) in RPMI medium (GIBCO) at 37 C for 1.5 hours before straining through a 70 mm cell filter (BD) and centrifugation (600 x g, 20 min) over an Optiprep (Axis-Shield) density gradient. Matched peripheral blood samples were obtained and peripheral blood mononuclear cells were separated by centrifugation over Lymphoprep (Axis-Shield) at 600 x g for 30 min.
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