HK-2 cells (human kidney proximal tubular cells) were cultured in Dulbecco’s modified Eagle’s medium/F12 medium (Life Technologies, Carlsbad, CA), which contains 5% FBS (Invitrogen) and 1% antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin) (Life Technologies). The cells were incubated at 37 °C in a humidified incubator with 5% CO2. To over-express or down-regulate the expression of specific miRNAs, cells were transiently transfected with miRNA mimics or inhibitor (Life Technologies) at 100 nM concentrations by using the Lipofectamine 3000 (Invitrogen) for indicated time points, according to the manufacturer’s instructions. The negative control contained a scrambled sequence. For TGF-β-treated experiment, cells were cultured in serum-free medium in the presence or absence of 5 ng/mL recombinant human TGF-β1 (R&D Systems, Minneapolis, MN) for different time points.
Dulbecco s modified eagle s medium f12 medium
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
Dulbecco's modified Eagle's medium/F12 medium is a cell culture medium commonly used for the growth and maintenance of a variety of cell types, including mammalian cells. It is a mixture of essential nutrients, vitamins, and other components required for cell proliferation and survival.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (14 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (4 mentions)
Epidermal growth factor (egf), by Merck Group (4 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (4 mentions)
B27 supplement, by Thermo Fisher Scientific (4 mentions)
Basic fibroblast growth factor (bfgf), by Merck Group (3 mentions)
Penicillin streptomycin, by Merck Group (3 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Recombinant human tgf β1, by R&D Systems (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Fibroblast growth factor 2 (fgf2), by Fujifilm (2 mentions)
Chromium single cell 3 reagent kit v2, by 10x Genomics (2 mentions)
B27 supplement minus vitamin a, by Thermo Fisher Scientific (2 mentions)
Bioanalyzer high sensitivity chip, by Agilent Technologies (2 mentions)
Dulbecco s modified eagle s medium dmem f12, by Thermo Fisher Scientific (2 mentions)
Kapa library quantification kit, by Roche (2 mentions)
Epidermal growth factor (egf), by Fujifilm (2 mentions)
56 protocols using dulbecco s modified eagle s medium f12 medium
1
Modulating miRNAs in HK-2 Cells
2
Establishing and Characterizing Glioblastoma Stem-Like Cells
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The establishment and characterization of glioblastoma stem-like cells (GSCs) have been previously reported (49 (link)). Briefly, GSCs were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Life Technologies) containing a B27 supplement minus vitamin A (Life Technologies), epidermal growth factor, and fibroblast growth factor 2 (20 ng/ml each; Wako Pure Chemicals Industries). For in vitro differentiation, GSCs were cultured in Dulbecco’s modified Eagle’s medium/F-12 medium (Life Technologies) containing 10 foetal bovine serum for the indicated times. Single-cell suspensions of GSCs or serum-induced differentiated GSCs were subjected to droplet-based scRNA-seq library preparation with the Chromium Single Cell 3’ Reagent Kit v2 (10× Genomics), aiming for an estimated 2,000 cells per library and following the manufacturer’s instructions. The libraries were checked with a BioAnalyzer High Sensitivity Chip (Agilent), quantified with a KAPA Library Quantification Kit (Roche), and then sequenced on the Illumina HiSeq 2500 platform in rapid mode.
3
Pancreatic Tumor Cell Culture Protocol
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Tumor cells were cultured from a primary pancreatic tumor isolated from a 17 week old RT2 AB6F1 male mouse; this mouse showed liver metastasis at the time of euthanasia. To deter growth of normal fibroblasts, the tumor cells were grown in a stem cell medium for several weeks, consisting of Dulbecco's modified Eagle's medium/F12 medium (Life Technologies) supplemented with N-2 supplement (Life Technologies), 20 ng/ml human epidermal growth factor (Life Technologies), 10 ng/ml human basic fibroblast growth factor (Sigma-Aldrich), 4 μg/ml heparin (Sigma-Aldrich), 4 mg/ml bovine serum albumin (Life Technologies), 20 μg/ml human insulin, zinc solution (Life Technologies), and 2.9 mg/ml glucose (Sigma-Aldrich) [46 (link)]. After several passages, the cells were switched to RPMI media with 10% fetal bovine serum. SV40 T-antigen antibody (Abcam), insulin antibody (Cell signaling) and CD88 antibody (Biolegend) were used for flow cytometry analysis. Invasiveness was tested using a matrigel invasion chamber (Corning) with or without addition of recombinant mouse C5a protein (R&D Systems). The invasion assay was repeated three times.
4
Cell Culture Protocol for Multiple Cell Lines
5
Isolation and Spheroid Culture of Tumor Cells
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Tumors were mechanically dissected into small pieces and enzymatically digested at 37 °C for 1 h into single-cell suspensions using collagenase A (50 U/mL, Roche, Basel, Switzerland) contained in Ca/Mg-free phosphate-buffered saline. Cells were incubated with Ber-EP4-coated magnetic Dynabeads (Life Technologies, Grand Island, NY) for 30 min to select epithelial cells, which were then cultured in RPMI medium (Gibco/Life Technologies, Grand Island, NY) containing 10% fetal bovine serum, 1% penicillin-streptomycin, and 20 ng/mL epidermal growth factor (Life Technologies).
For spheroid formation, single cells were plated on ultra-attachment six-well culture plates (Corning, Acton, MA) at a density of 1 × 10^3 cells/cm2 in serum-free Dulbecco’s modified Eagle’s medium/F12 medium (Life Technologies) supplemented with 20 ng/mL epidermal growth factor (Life Technologies), 10 ng/mL basic fibroblast growth factor (Sigma-Aldrich), and 5 μg/mL insulin (Sigma-Aldrich). Spheroid formation of 50–100 cells was assessed at 7 days after seeding.
For spheroid formation, single cells were plated on ultra-attachment six-well culture plates (Corning, Acton, MA) at a density of 1 × 10^3 cells/cm2 in serum-free Dulbecco’s modified Eagle’s medium/F12 medium (Life Technologies) supplemented with 20 ng/mL epidermal growth factor (Life Technologies), 10 ng/mL basic fibroblast growth factor (Sigma-Aldrich), and 5 μg/mL insulin (Sigma-Aldrich). Spheroid formation of 50–100 cells was assessed at 7 days after seeding.
6
Establishing Xenograft Models of Liposarcoma
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Female adult, partially immunodeficient, athymic NMRI nu/nu mice (JANVIER LABS, Saint Berthevin, France) were used for establishing xenograft models and for the in vivo experiments. Collection and usage of tumor samples from consenting patients were approved by the Medical Ethics Committee, University Hospitals Leuven. Animal experiments were approved by the Ethics Committee for Laboratory Animals, KU Leuven (Leuven, Belgium).
The SW872 liposarcoma cell line (Cell Lines Service, Eppelheim, Germany) was cultured in Dulbecco's modified Eagle's medium/F12 medium with 10% FBS (all from Life Technologies). The SW872 cell line has been previously studied in vitro and in vivo[15] (link), [16] (link). The SW872 model was generated by subcutaneous, bilateral injection of 5 × 106 cells per mouse site. Patient-derived DDLPS xenografts (UZLX-STS3 and UZLX-STS5) were established by bilateral subcutaneous implantation of fresh surgically resected tumor specimens from patients with DDLPS. Tumor tissue was further re-transplanted from mouse to mouse at least twice. From each passage, tumor fragments were collected for histologic and molecular characterization.
The SW872 liposarcoma cell line (Cell Lines Service, Eppelheim, Germany) was cultured in Dulbecco's modified Eagle's medium/F12 medium with 10% FBS (all from Life Technologies). The SW872 cell line has been previously studied in vitro and in vivo[15] (link), [16] (link). The SW872 model was generated by subcutaneous, bilateral injection of 5 × 106 cells per mouse site. Patient-derived DDLPS xenografts (UZLX-STS3 and UZLX-STS5) were established by bilateral subcutaneous implantation of fresh surgically resected tumor specimens from patients with DDLPS. Tumor tissue was further re-transplanted from mouse to mouse at least twice. From each passage, tumor fragments were collected for histologic and molecular characterization.
7
Cell Culture Protocols for Diverse Cell Lines
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HeLa cells (American Type Culture Collection; ATCC) and rat embryonic fibroblasts stably expressing YFP-Paxillin (REF-Pax32 (link)) were grown in high-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM L -Glutamine, 10% FBS, 1% penicillin/streptomycin (all Sigma-Aldrich). Human Umbilical Vein Endothelial Cells (HUVECs, Life Technologies) were grown in M200 medium with Low Serum Growth Supplement (all Life Technologies). MCF10A cells (G. Scita, IFOM Milan, Italy) were grown in Dulbecco's modified Eagle's medium /F12 medium (Life Technologies) supplemented with 10% horse serum, 1% penicillin/streptomycin, 0.5 mg ml−1 hydrocortisone, 10 μg ml−1 insulin, 20 ng ml−1 epidermal growth factor (all Sigma-Aldrich) and 100 ng ml−1 Cholera toxin (List Biological Laboratories). All cells were cultured in a humid atmosphere containing 5% CO2 at 37 °C.
PC12 cells (ATCC) were grown in RPMI-1640 medium supplemented with 10% horse serum, 5% FBS, 2 mML -glutamine and 1% penicillin/streptomycin (all Sigma-Aldrich). Details on the cytotoxicity test are described elsewhere33 (link). Briefly, PC12 cells were grown for 4 days in RPMI-1640 medium supplemented with 2% FBS, 2mM L -glutamine, 1% penicillin/streptomycin and 100 ng ml−1 nerve growth factor (induction medium), followed by measuring the mean neurite length.
PC12 cells (ATCC) were grown in RPMI-1640 medium supplemented with 10% horse serum, 5% FBS, 2 mM
8
Modulation of Renal Fibrosis Factors by miR-1470 and miR-4483
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HK-2 cells were cultured in Dulbecco's modified Eagle's medium/F12 medium(Life Technologies, Carlsbad, CA) with 10% fetal bovine serum (FBS), 100-ug/mL penicillin and streptomycin (Life Technologies) at a 37 °C incubator with 5% CO2. In the six-well plate, in line with the instructions of ribo FECTTMCP transfection reagent,50 nM miR-1470, miR-4483 mimics and mimic negative control, 100 nM miR-1470, miR-4483 inhibitor and inhibitor negative control were transfected into HK-2 cells respectively, and the transfected cells were obtained at the designated time point to detect microRNA levels and expression of fibrosis factors. (The sequence of microRNA mimic, inhibitor, mimic NC and inhibitor NC are shown in Supplementary Table 3.) For the TGF-β1 or high glucose (HG) treated experiment, cells were cultured in the presence or absence of 10 ng/mL recombinant human TGF-β1 (R&D Systems, MN) or 30 mM HG for various times.
9
Evaluating AMSC and Wil Fibrils
10
Culturing Mouse Microglial BV-2 Cells
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The mouse microglial cell line BV-2 was generated from primary mouse microglia transfected with a v-raf/v-myc oncogene and was maintained at 37∘C in Dulbecco’s modified Eagle’s medium/F12 medium (Life Technologies, Grand Island, NY, United States) with 10% heat-inactivated FBS (HyClone, Logan, UT, United States), 50 U ml–1 penicillin, and 50 mg/ml streptomycin (Sigma Chemical, St. Louis, MO, United States) under a humidified atmosphere of 5% CO2 and 95% air. Cell numbers seeded 1 × 106 cells/dish in 10 cm dish for Western blot analysis and quantitative PCR (qPCR) analysis.
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