After removal of the olfactory bulb and cerebellum, brains were cut into small 1–5 mm pieces in DMEM containing 1 mg/ml collagenase A (Roche) and 0.01 mg/ml deoxyribonuclease type I (Sigma) and incubated for 30 min at 37°C. Tissue fragments were dissociated using a 40 μm cell strainer (EASY strainer, GBO). Kidneys were cut with into small 1–5 mm fragments in DMEM containing 10% fetal calf serum, 0.4 mg/ml collagenase D (Roche) and 0.02 mg/ml deoxyribonuclease type I (Sigma) and dissociated at 37°C for 30 min using a GentleMACS apparatus (Miltenyi Biotec). Cell pellets were gently resuspended and layered onto a 30% (brain cells) or 40% (kidney cells) Percoll gradient (GE healthcare) and bands corresponding to leucocytes were collected. Spleens were minced in DMEM, 10% FCS using a 70 μm cell strainer (EASY strainer, GBO). Cells were washed and red-blood-cells were lysed using ACK erythrocyte lysis buffer (155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH 7.2). After washing, cells were resuspended in FACS Buffer.
Deoxyribonuclease type 1
Manufactured by Merck Group
Deoxyribonuclease type I is a laboratory enzyme that catalyzes the hydrolysis of DNA. It is used to break down DNA molecules into smaller fragments in various biochemical and molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Collagenase type 4, by Merck Group (2 mentions)
Facsaria 2 instrument, by BD (1 mentions)
Fc block, by BD (1 mentions)
Cytofix cytoperm buf kit, by BD (1 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
Collagenase 1, by Merck Group (1 mentions)
Percoll gradient, by GE Healthcare (1 mentions)
Collagenase d, by Roche (1 mentions)
Collagenase a, by Roche (1 mentions)
Collagenase type ia, by Merck Group (1 mentions)
Facsaria 2 cell sorter, by BD (1 mentions)
Gentamycin, by Merck Group (1 mentions)
Trypsin inhibitor, by Merck Group (1 mentions)
Bovine serum albumin fraction 5, by Merck Group (1 mentions)
Neurobasal a medium, by Thermo Fisher Scientific (1 mentions)
B27 supplement, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Merck Group (1 mentions)
Poly l ornithine, by Merck Group (1 mentions)
Kynurenic acid, by Merck Group (1 mentions)
8 protocols using deoxyribonuclease type 1
1
Isolation and Purification of Organ-Specific Cells
2
Isolation of Hepatic Macrophages
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The left lobes of the livers were gently lysed and digested for 20 min at 37 °C using type IV collagenase (Sigma-Aldrich) and type I deoxyribonuclease (Sigma-Aldrich) in phosphate-buffered saline containing 2% bovine serum albumin (pH 7.4). Non-parenchymal cells were incubated with Fc Block (BD Biosciences, San Jose, CA) and then with fluorochrome-conjugated antibodies (Supplemental Table 2 ). Cells were analyzed using a FACSAria II instrument (BD Bioscience), as described previously43 (link). Hepatic macrophages were identified as propidium iodine−NK1.1−CD3−CD19−TER119−CD45+CD11b+F4/80+ cells. Data analysis was performed using FlowJo software (Tree Star, Ashland, OR).
3
Comprehensive Tumor-Immune Cell Characterization
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For cell surface staining, cell suspensions were washed twice in PBS and stained with indicated fluorescent labelled antibodies for 30 min on ice and washed with PBS. For TILs isolation, tumours were cut, minced, followed by incubation with Type IV Collagenase(Sigma) and Type I Deoxyribonuclease (Sigma) for 60 min at 37 °C with gentle shaking. After passing through a 70-µm filter, lymphocytes were purified from the interface of mouse Ficoll gradient centrifugation (MultiSciences). For intracellular staining, the cells were sorted for fixation and permeabilization using the Cytofix/CytoPerm buf kit (Cat# 554714, BD Bioscience). For detecting the proliferation of T-cell, human HLA-A2+ T lymphocytes were labelled with CFSE, and activated in vitro with anti-CD3 pre-coated U-96-plates in present of 100 U/ml IL2. When needed, irradiated (100 Gy) breast cancer cells were added in co-culture system at the indicated ratio in triples. All flow cytometry analysis were conducted on CytoFlex (Beckman) and the data were analyzed using FlowJo software according to the manufacturers’ instructions. Compensation beads were used to evaluate spectral overlap, compensation was automatically calculated. All antibodies used for flow cytometry analysis are listed in Supplementary Table 3 .
4
Isolation and Culture of Endometrial Stem Cells
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One gram of endometrial tissue was stripped from the underlying myometrium and dissociated using mechanical and enzymatic digestion as previously described with a few modifications. Briefly, the tissue pieces were washed twice in Phosphate Buffered Saline (PBS) and minced before dissociation in DMEM/F-12 containing 0.1% Bovine Serum Albumin (BSA), 0.5% collagenase I, 40 μg/mL deoxyribonuclease type I (Sigma Aldrich), and 1% penicillin/streptomycin for 40 min at 37 °C in a SI50 Orbital Incubator (Stuart Scientific). The resulting cell solution was filtered through a sterile 70-μm cell strainer (Fisher Scientific) to separate single cells from undigested tissue fragments following isolation. Patient endometrial SCs (EuSCs, EcSCs, and control cells) were then cultured at 37 °C and 5% CO2 in DMEM supplemented with 10% heat-inactivated fetal bovine serum. Cells were passaged upon reaching 90% confluence.
5
Isolation of Prostate-Infiltrating Neutrophils
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Ventral prostates from rats with LPS-induced prostatitis were quickly excised, rinsed in fresh saline and weighted. Cell dissociation and neutrophil purification were performed using an adaptation of a published protocol (26 (link)). Briefly, tissues were minced into small fragments and treated with a digestion solution containing 200 U/ml collagenase type IA (Sigma Aldrich, St. Louis, MO) and 0.05% deoxyribonuclease type I (Sigma Aldrich, St. Louis, MO) in HBSS without calcium, magnesium or phenol red, pH 7.4 for 30 min, at 37°C, and rocking at 60 rpm. The digested tissue was then passed through a 70-μm pore cell strainer with fresh sterile PBS. The cell suspension was then spun for 6 min, 300 × g at 20°C. After removal of the supernatant, residual red blood cells were removed by hypotonic lysis and cells were spun again for 6 min, 300 × g, at 20°C, washed, and resuspended in 1 ml of PBS. Cells were counted by collecting events for a fixed time (90 s) on a FACSCanto II cytometer. Neutrophil percentage was calculated by using a FITC anti-rat Gr monoclonal antibody (1/500 for 1 h at 4°C).
To purify prostate-infiltrating neutrophils, Gr (+) cells were sorted in a FACSAria II cell sorter (BD Biosciences, San Jose, CA), with a purity of >97% being achieved and confirmed by electron microscopy.
To purify prostate-infiltrating neutrophils, Gr (+) cells were sorted in a FACSAria II cell sorter (BD Biosciences, San Jose, CA), with a purity of >97% being achieved and confirmed by electron microscopy.
6
Hippocampal Primary Neuron Culture Protocol
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Hippocampal primary cultures were prepared as previously described (33 (link)). Briefly, hippocampi isolated postnatal day 0 to 3 Wistar rats were digested by three separate enzymatic [trypsin (5 mg/ml; Sigma-Aldrich, no. T10051), deoxyribonuclease type I (0.75 mg/ml; Sigma-Aldrich, no. D5025-150KU), trypsin inhibitor (2.5 mg/ml; Sigma-Aldrich, no. T9003)] and mechanic steps alternated with washings [4.2 mM NaHCO3, 12 mM Hepes (Merck, no. 4034), 200 μM kynurenic acid (Sigma-Aldrich, no. K3375), 25 μM D-AP5 (Abcam, no. ab120003), powder Hank’s modified (Sigma-Aldrich, no. H-2387), 33 mM glucose, bovine serum albumin (BSA) fraction V (0.3 mg/ml; Merck, no. 10735086001), 12 mM MgSO4, and gentamycin (0.25 μl/ml; Sigma-Aldrich, no. G12)]. The final pellet of cell was resuspended in Neurobasal medium [1× Neurobasal-A Medium (Gibco, no. 10888-022) supplemented with 1× GlutaMAX (Gibco, no. 35050-061), 1× B27 supplement (Life Technologies, no. 17504044), and gentamycin (0.5 μl/ml; Sigma-Aldrich, no. G1272)]. Cells were then plated on a 12 mm–by–24 mm coverslip (150,000 cells per coverslip) pretreated with poly-l -ornithine (Sigma-Aldrich, no. P4957) and maintained (7 to 10 days in vitro) in an incubator at 37°C and 5% CO2.
7
Neonatal Mouse Brain Cell Culture
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Neonatal C57BL6 mice were sacrificed by decapitation. Brains were dissected and the olfactory bulb, cerebellum and meninges were removed under the microscope. A cell suspension was prepared by mincing brain tissue in Hank’s balanced salt solution. Cell pellets were collected and treated with 0.5 mg/ml papain (Sigma) and 0.01 mg/ml deoxyribonuclease type I (Sigma) in Hank’s balanced salt solution, for 30 min at 37°C. Cells were then resuspended with BME medium supplemented with 10% FCS and filtered through a 70 μm cell strainer (EASY strainer, GBO). Cell pellets were collected, resuspended in 10% FCS BME medium and cultured in T-75 flasks (Sigma) for 21 days at 37°C with 5% CO2.
8
Isolation and Characterization of Human Endometrial Stromal Cells
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Human endometrial tissue was washed in phosphate-buffered saline and then minced in a medium containing Dulbecco Modified Eagle Medium/Ham's F-12 (DMEM/F-12) supplemented with 100 mg/mL penicillin and 100 mg/mL streptomycin. The tissue fragments were digested with collagenase type 3 (300 μg/mL; Sigma) and deoxyribonuclease type I (40 μg/mL; Sigma) at 37 °C for 90 minutes, and then dissociated into a single cell by vortexing briefly. After removal of the glandular and epithelial components, the cell suspension passed sequentially through meshes of 100, 70, and 40 sieves (BD Biosciences), respectively. Cell viability and numbers were assessed by trypan blue staining. The purified endometrial stromal cells were seeded at 2 × 105 cells per well of 6-well cell culture plates in DMEM/F-12 supplemented with penicillin and streptomycin (Invitrogen) and 5% fetal bovine serum (FBS), then they were incubated in 5% CO2 at 37 °C. The medium was changed every 2 to 3 days until they reached 80% to 100% confluency. The stromal cell identity was confirmed using Vimentin staining in confluent cells (>99% by our estimation) by immunohistochemical analysis as described previously.
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