Tumor tissue and lymph nodes were cut into pieces of <1 mm3 and placed in a T75 culture flask (Nunc™ EasYFlask™ Cell Culture Flasks, cat. no. 156499, ThermoScientific) with digestion medium, consisting of RPMI (Gibco, Paisley, UK), 10% fetal bovine serum (FBS, Gibco, Paisley, UK), collagenase type IV (1 mg/mL; Gibco, Grand Island, USA), and 12.6 µg/mL recombinant human DNase (Pulmozyme, Roche, Woerden, the Netherlands) for overnight digestion at room temperature. After digestion, the suspension was strained through a 70 µm filter and washed with PBS. Cells were centrifuged over a Ficoll-Paque gradient (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and lymphocytes were isolated from between the two layers. After a wash with PBS, cells were pelleted. Total cell pellet was suspended in 1 ml FBS with 10% dimethylsulfoxide (Merck, Darmstadt, Germany), and stored in liquid nitrogen until further use. Peripheral blood was centrifuged over a Ficoll-Paque gradient (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and PBMC were isolated from between the two layers. After a wash with PBS, cells were pelleted. Total cell pellet was suspended in 1 ml FBS with 10% dimethylsulfoxide (Merck, Darmstadt, Germany), and stored in liquid nitrogen until further use.
Recombinant human dnase
Manufactured by Roche
Sourced in United States
Recombinant human DNase is a laboratory enzyme used to degrade DNA. It is produced using recombinant DNA technology.
Automatically generated - may contain errors
Lab products found in correlation
Collagenase type 4, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Rpmi medium, by Thermo Fisher Scientific (2 mentions)
Ficoll paque plus, by GE Healthcare (2 mentions)
70 m cell strainer, by BD (2 mentions)
Ficoll paque gradient, by GE Healthcare (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions)
4 protocols using recombinant human dnase
1
Isolation and Cryopreservation of Tumor and Peripheral Lymphocytes
2
Tumor Tissue Isolation and Cryopreservation
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Tumor tissue from endometrial carcinoma patients was collected during primary surgery. Written informed consent was obtained from all patients. Tumor molecular analysis on mismatch repair (MMR) protein expression was performed as part of standard-of-care. Tumors were minced into small pieces and enzymatically digested using 1 mg/µL collagenase type IV (Gibco Life Technologies, Grand Island, NE, USA) and 12.6 µg/mL recombinant human DNase (Pulmozyme, Roche, Woerden, The Netherlands) in RPMI medium (Gibco, Paisley, UK). Digestion was either done overnight at room temperature or for 30 min at 37 °C. Digests were filtered using 70 µm cell strainers (Falcon) and enriched for mononuclear cells using Ficoll-Paque PLUS (GE Healthcare Life Sciences, Marlborough, MA, USA). After washing, cells were cryopreserved in fetal bovine serum (FBS, Gibco, Paisley, UK), with 10% dimethylsulfoxide until used for experiments.
3
Competitive DNase Activity Assay
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In brief, a competitive DNase assay was established using chicken antibodies (NABAS, Ås, Norway) as a coat and recombinant human DNase (Roche, Basel, Switzerland) that was conjugated with HRP and mixed with a sample to be examined for readout. Readings at 450 nm after the addition of substrate were inversely proportional to the DNase concentration in the sample, expressed in ng/mL [26 (link)].
4
Endometrial Cancer Tissue Digestion
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Endometrial cancer digests were obtained from surgical waste material collected in the University Medical Center Groningen, The Netherlands. Patients had given consent to use surgical waste material for research purposes. Samples were processed anonymously and assigned a coding number prior to storage. According to Dutch law no approval from our institutional review board was needed. Of the material used for sequencing, two tumors were high-grade endometrioid adenocarcinomas with MMR deficiency (FIGO IA, loss of PMS2, and FIGO IB, loss of MSH2 and MSH6, respectively) and the third tumor was of high-grade serous p53-mutant adenocarcinoma histology with proficient mismatch repair proteins, FIGO IIIC2. Tumors were cut into approximately 1cm3, enzymatically digested in RPMI medium (Gibco, Paisley, UK) with 1 mg/µL collagenase type IV (Gibco Life Technologies, Grand Island, NY, USA) and 12.6 µg/mL recombinant human DNase (Pulmozyme, Roche, Woerden, the Netherlands) for 30 min at 37 °C or overnight at room temperature. Digests were filtered using 70 µm cell strainers (Falcon) and enriched for peripheral blood mononuclear cells (PBMCs) using Ficoll-Paque PLUS (GE Healthcare Life Sciences, Marlborough, MA, USA). Cells were cryopreserved in fetal calf serum (FCS) with 10% dimethylsulfoxide and stored in liquid nitrogen until further use.
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