Blood samples were collected from 20 mBC patients included in the study before and 3-4 weeks after starting the treatment and from 5 healthy women as controls. Peripheral blood mononuclear cells (PBMCs) were freshly isolated (within 5 hours after blood drawing) from blood samples collected in EDTA before therapy by Ficoll-Hypaque gradient (Lymphoprep, Fresenius Kabi Norge Halden) using standard gradient separation. Cells were washed in PBS (Biomerieux), counted using an automated cell counter (ADAM-MC™, DigitalBio, NanoEnTek Inc.) and viably frozen [90% heat-inactivated Fetal Bovine Serum (FBS; Euroclone) and 10% DMSO] at -80°C for 24 h and then in liquid nitrogen until use. After thawing in IMDM (Lonza) containing 2 mM L-glutamine, 100 μg/ml streptomycin and 100 IU/ml penicillin (Sigma-Aldrich), supplemented with 2% human serum (Sigma-Aldrich) and with 3 μg/ml Deoxyribonuclease (Sigma-Aldrich), cells were washed in PBS (Biomerieux) and counted again to check viability (>80%). Five hundred µl buffy coat samples were obtained from 7.5 ml EDTA-blood samples collected after therapy and centrifuged at 3600 rpm for 10 min, then maintained at -80°C until use.
Deoxyribonuclease
Manufactured by Merck Group
Sourced in United States
Deoxyribonuclease is an enzyme that catalyzes the hydrolytic cleavage of phosphodiester linkages in the backbone of DNA molecules, resulting in the breakdown of DNA. It is a commonly used tool in molecular biology and biochemistry laboratories.
Automatically generated - may contain errors
Lab products found in correlation
Hyaluronidase, by Merck Group (12 mentions)
Collagenase 4, by Merck Group (6 mentions)
Collagenase, by Merck Group (6 mentions)
Collagenase type 4, by Merck Group (4 mentions)
Collagenase 3, by Worthington (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Moflo machine, by Beckman Coulter (2 mentions)
Hank s balanced salt solution, by Thermo Fisher Scientific (2 mentions)
Collagenase b, by Roche (2 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (2 mentions)
Viability dye, by Thermo Fisher Scientific (2 mentions)
Lsrii flow cytometry machine, by BD (2 mentions)
Software, by FlowJo (2 mentions)
Protease, by Merck Group (2 mentions)
Penicillin streptomycin, by Merck Group (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Human serum, by Merck Group (1 mentions)
Iscove modified dulbecco media (imdm), by Lonza (1 mentions)
47 protocols using deoxyribonuclease
1
Peripheral Blood Immune Profiling in mBC
2
Immune Response Signaling Pathway Modulation
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The TLR agonists Pam3Cys, polyI:C, LPS, flagellin, and R848 were purchased from Invivogen (San Diego, CA, USA); CpG-2006 and CpG-1826 from Invitrogen (Carlsbad, CA, USA) or Genomics Biosci & Tech (New Taipei, Taiwan); BMS-345541, collagenase II, collagenase IV, deoxyribonuclease, and anti-Flag M2 antibody from Sigma-Aldrich Co. (St. Louis, MO, USA); and HA.11, PE-anti-human CD117, PE- anti-mouse CD117, and PE-rat IgG2b κ isotype control antibody from BioLegend (San Diego, CA, USA); PE-anti-human CD133 antibody from Miltenyi Biotec (Bergisch Gladbach, Germany); PE-anti-mouse CD11b antibody from eBioscience (San Diego, CA, USA); anti-phospho-RelA (Alexa Fluor 647 conjugate) and rabbit IgG isotype control antibody from Cell Signaling (Beverley, MA, USA). The miR-205 mimics and control were synthesized by GeneDirex (Gueishan Township, Taiwan). Antibodies against human and mouse COMMD1 were purchased from Abnova (Taipei, Taiwan) and Proteintech (Chicago, IL, USA), respectively; human recombinant TNF-α, IL-1β, epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF) from Peprotech (Rocky Hill, NJ, USA); and reagents for luciferase assay, from Promega (Medison, WI, USA).
3
Isolation of Renal Progenitor Cells
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Renal progenitor cells were isolated from the renal cortex of model and sham mice after 16-h RIRI surgery. Kidney was perfused via the aorta with the buffer (50 mM PBS and 80 U/mL heparin). Renal capsules were removed by using forceps. Isolated renal tissues were immersed in ice-cold DMEM medium. The tissues were was sliced and homogenized into 1 mm3 pieces, and resuspended in Collagenase type IV solution (Collagenase type IV 1 mg/mL), Deoxyribonuclease 0.1 mg/mL, Bovine serum albumin (BSA) 1 mg/mL, and all regents were from Sigma Chemical Co (St. Louis, MO, USA). The mixture was cultured at 37 °C in with 100-rpm shaking for 15 min. The mixture was homogenized by pipetting ten times through a sterile transfer pipette. One-milliliter Collagenase type IV solution was added and whole procedure was repeated three times. A total of 20-mL DMEM medium was added into the digestive solution. The suspension was centrifuged at 200 g for 5 min. The pellets were resuspended and washed by DMEM medium for three times. Renal progenitor cells were collected via density-gradient centrifugation of 45% (v/v) sterile Percoll solution. Isolated cells were washed three times with cold DMEM for next step.
4
Immune Cell Isolation Protocol
5
Tumor-Infiltrating Immune Cell Isolation
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Cell isolation was performed as previously described by VanValkenburgh et al. (32 (link)). For isolation of immune cells from B16-Ova flank tumors, tumors were excised from mice, and tissue was homogenized using the GentleMacs system on m.Tumor.01 (Miltenyi). Tissue was further incubated with collagenase D (2.0 mg/ml; Worthington) and 7.5 of μl deoxyribonuclease (Sigma-Aldrich) for 30 min at 37°C in a shaking incubator. Samples were passed through a 70-μm filter, and tumor cells were removed by a 44/66% Percoll gradient centrifugation. For intracellular and TF staining, the eBioscience Foxp3/Transcription Factor Staining Buffer Set (cat. no. 00-5523-00) was used. Flow cytometry was performed on a BD LSR II, data acquisition was performed on BD FACSDiva Software, and flow cytometry files were analyzed on FlowJo. Antibodies are listed in table S4.
6
Decellularized ECM from Human MSCs
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Human bone marrow–derived MSCs (Lonza, Walkersville, MD) were used without further characterization. MSCs were expanded until use at passages 5 and 6 in minimum essential medium–α [α-MEM; with l -glutamine and without ribo/deoxyribonucleosides (Invitrogen, Carlsbad, CA)] supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals, Flowery Branch, GA) and 1% penicillin (10,000 U ml−1) and streptomycin (10 mg ml−1; Mediatech, Manassas, VA) (P/S). Cell-secreted ECMs were prepared as we described (28 (link), 35 (link), 45 (link), 46 (link)). Briefly, MSCs were seeded at 50,000 cells cm−2 and cultured in medium supplemented with ascorbate 2-phosphate (50 μg ml−1) for 10 days with medium changes every 2 to 3 days. After culture, monolayers were washed with phosphate-buffered saline (PBS), and cells were removed using a detergent-based solution, followed by deoxyribonuclease (Sigma-Aldrich, St. Louis, MO) treatment (37°C for 1 hour) to remove 99.9% of DNA content from culture after decellularization (45 (link)). Decellularized ECM was washed three times with PBS and mechanically dislodged from culture flasks using a cell scraper. Total protein within the collected ECM was quantified using a bicinchoninic acid protein assay (Thermo Fisher Scientific, Rockford, IL). ECM solutions were frozen in 0.02 N acetic acid at −20°C until use.
7
Quantification of Interferon and IGFBP1 Expression
8
Isolation and Expansion of Tumor-Infiltrating T Cells
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BC PDXs were minced into small pieces and digested with a triple-enzyme mixture containing collagenase type IV (0.5 mg/mL; Sigma-Aldrich), hyaronidase (0.5 mg/mL; Sigma-Aldrich), and deoxyribonuclease (0.02 mg/mL; Sigma-Aldrich) for 120 min at room temperature. After digestion, BC cells were washed twice in PBS and cultured in RPMI-1640 containing 10% human serum supplemented with l -glutamine (Sigma-Aldrich), 2-mercaptethanol (Sigma-Aldrich), and IL-2 (1000 U/mL; Cat# 130-097-742; Miltenyi Biotec) for T cell generation. Following release of T cells from tumor tissues, they were grown in high-dose IL-2 (1000 U/mL)-containing medium for 1 week, followed by transfer to a fresh well and growth in low-dose IL-2 (50 U/mL)-containing medium.
9
Isolation and Characterization of Immune Cells
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The tissue of transplanted area was minced with a scissors, after which the tissue fragments were incubated for 1 h at 37 ºC in Dulbecco's Modified Eagle Medium (DMEM: Invitrogen) in the presence of 0.2% collagenase (Wako Chemicals, Cell dissociation grade), and 25 μg/ml deoxyribonuclease (Sigma-Aldrich). The suspension was filtered through a cell strainer (Falcon 2350, 70 μm) to remove debris and tissue fragments, after which the cells were pelleted by centrifugation at 300 g for 5 min at room temperature. Next, the cells were resuspended in calcium- and magnesium-free Hank's Balanced Salt Solution (HBSS) (Fujifilm) supplemented with 2% FBS, 10 mM HEPES and 1% penicillin/streptomycin (P/S). The cells were stained with immune cell markers as described below; PE conjugated mouse anti-rat CD3 (BD Pharmingen, Clone G4.18, Catalog No:554833), FITC conjugated mouse anti-rat CD4 (BD Pharmingen, Clone OX-35, Catalog No:554837), Catalog No:561588), PE conjugated mouse anti-rat CD11b/c (BD Pharmingen, Clone OX-42, Catalog No:554862), and Alexa Fluor® 647 conjugated mouse anti-rat CD163 (Bio-Rad Laboratories, Inc., Clone ED2, Catalog No: MCA342A647). Flow-cytometric analysis was performed on FACSMelody (BD Biosciences), and the data were analyzed using FlowJo software (BD Biosciences).
10
Isolation and Activation of Hepatic Stellate Cells
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HSCs were isolated from adult Sprague-Dawley (SD) rats (8 weeks of age) or mice and primary rat HSCs were isolated using a two-step collagenase perfusion. Briefly, the rat liver was minced and incubated in 0.02% pronase with deoxyribonuclease (Sigma) after the rat liver perfused in situ with collagenase (Sigma) and streptavidin (Sigma). Then, the mixture was centrifuged to remove parenchymal cells and subsequently HSCs were recovered by density gradient centrifugation of nonparenchymal cells. HSCs were activated for 10–14 days by culturing in a 25 cm2 flask with DMEM (Gibco) containing 10% fetal bovine serum (Gibco) at 37°C/5% CO2 fully humidified atmosphere. The activated HSCs between passages 3 and 8 were used for experiments.
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