Human monocyte-derived DCs (moDCs) were differentiated from buffy coats of healthy donors according to Sender et al.50 (link). Briefly, human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll (Histopaque 1077, Sigma Aldrich, Steinheim, Germany) density gradient centrifugation from buffy coats (commercially obtained from: Blutspendedienst, Frankfurt am Main, Germany). Purification of monocytes was performed by positive selection using CD14 MicroBeads (Miltenyi Biotech). For generation of moDCs, 1 × 106 cells/ml were seeded in 24-well plates (Thermo Scientific) in complete medium (RPMI 1640 (Sigma-Aldrich), 10% FCS, 10 mM HEPES, 2 mM L-Glutamine (Biochrome), 100 U Penicillin/Streptomycin (Biochrome), 25 mM beta-mercapto ethanol) supplemented with 5 ng/ml GM-CSF (Leukine, Sanofi-Aventis, Frankfurt, Germany) and 10 ng/ml IL-4 (CellGenix, Freiburg, Germany). Cells were differentiated at 37 °C and 5% CO2 for five days. Human moDCs were harvested on day 5 and stimulated with the indicated, equimolar amounts of the different proteins for 24 h. 1 ng/ml LPS (Sigma Aldrich) served as positive control. Supernatants were analyzed for cytokine secretion by ELISA.
Penicillin streptomycin
Manufactured by Biochrome
Sourced in Germany
Penicillin/streptomycin is a mixture of two antibiotics commonly used in cell culture applications. It is designed to inhibit the growth of a wide range of bacteria, including both gram-positive and gram-negative species.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Biochrome (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Rpmi 1640, by PAN Biotech (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Vorinostat, by Selleck Chemicals (2 mentions)
Mithramycin a, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
24 well plate, by Thermo Fisher Scientific (1 mentions)
Interleukin 4 (il 4), by CellGenix (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Histopaque 1077, by Merck Group (1 mentions)
Cd14 microbead, by Miltenyi Biotec (1 mentions)
L glutamine, by Biochrome (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
6 well plate, by Corning (1 mentions)
Dulbecco modified eagle medium (dmem), by Biochrome (1 mentions)
17β estradiol, by Merck Group (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Tissuelyser 2, by Qiagen (1 mentions)
12 protocols using penicillin streptomycin
1
Generating Human Monocyte-Derived Dendritic Cells
2
Hypothalamic Neuronal Cell Line: AMPK Modulation
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The adult mouse derived hypothalamic neuronal cell line mHypoA-2/30 (CELLutions Biosystems Inc, Toronto, Canada), was used as an experimental model. These cells possess neuron-specific markers and function as effective models to study neuroendocrine signalling at the cellular level (24 (link)). Cells were grown in 6 well plates (Corning life Sciences, Flintshire, UK) with high glucose (HG) Dulbecco’s Modified Eagle’s Medium (DMEM, 4500 mg/L, Sigma-Aldrich, Dorset, UK) supplemented with 5% fetal bovine serum (FBS, Gibco, Paisley UK) and 1% penicillin-streptomycin (Biochrome AG, Berlin, Germany) maintained at 37°C and 5% CO2 until 90% confluency. A HG medium was used to reduce basal AMPK activity. In these HG conditions we explored effects of Ucn2 and AICAR on phospho-AMPK by Western Blot.
3
Estrogen Induction in Breast Cancer Cell Lines
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MCF7 cells were kindly provided by K. Effenberger (University Medical Center, Hamburg-Eppendorf) and were grown in phenol red-free high-glucose Dulbecco's modified Eagle's media (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (Thermo Scientific), 1% sodium pyruvate, and 1% penicillin/streptomycin (Sigma-Aldrich) at 37°C. For estrogen induction, cells were deprived of hormones by treating them with DMEM media supplemented with 5% charcoal-dextran-treated fetal bovine serum (CSS; Biochrome), 1% sodium pyruvate, and 1% penicillin/streptomycin after 24 hr of growth. After 48 hr, cells were treated with 10 nM 17 β-estradiol (Sigma-Aldrich) and/or 1 μM ICI-182,780 (Fulvestrant) for 2 hr. MCF10A cells were kindly provided by M. Oren (Weizmann Institute of Science, Israel) and were grown in phenol-red free DMEM/F12 medium supplemented with 5% horse serum, 20 ng/mL epidermal growth factor, 0.1 μg/mL Cholera toxin, 10 μg/mL insulin, 0.5 μg/mL hydrocortisone, 1% penicillin/streptomycin at 37°C.
4
Tissue Homogenization and RNA Extraction
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Organ pieces (approximately 2 × 2 × 2 mm) were transferred into 2 mL collection tubes prepared with a stainless-steel bead (diameter 5 mm) and 1 mL of DMEM supplemented with antibiotics (1% penicillin-streptomycin, Biochrome, Berlin, Germany). Homogenization was performed using a TissueLyser II instrument (Qiagen, Hilden, Germany) for 2 min at 300 Hz. Supernatants for RNA extractions were acquired following centrifugation at 13,000 rpm for 2 min.
5
Adipogenic Differentiation of Sorted Cells
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Sorted cells (Sca1(+), CD140a(–), CD31(–), CD45(–), CD11b(–)) were seeded at 104 cells cm–2 and cultured on Matrigel (Corning 356235)-coated 6-well plates in high-glucose DMEM (Thermo 11965092) supplemented with 5 ng mL–1 of bFGF (Sigma F3685), FBS 20% (GE SH30071), 2 mM of L-glutamine (Sigma G3126), Amphotericin B 1% (Sigma A2942), and penicillin/streptomycin 1% (Biochrome A2210). After stromal cell expansion, adipogenic differentiation was induced via reducing FBS to 5% and replacing bFGF with 0.5 mM of IBMX (Sigma I5879), 0.25 μM of dexamethasone (Sigma D4902), and 10 μg mL–1 of insulin (Sigma I1507), as described previously [20 (link)].
6
Characterization of MCC Cell Lines
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The classical MCPyV+ MCC cell lines BroLi, MKL-1, MKL-2, and WaGa have been described before51 . All cell lines were authenticated by STR analysis on regular bases (last performed in June 2016). Cell lines were maintained in RPMI-1640 (PAN Biotech, Aidenbach, Germany) supplemented with 10% fetal bovine serum (FBS; Biochrom, Berlin, Germany) and 1% penicillin/streptomycin (Biochrome, Berlin, Germany). For treatment with specific inhibitors, cells were cultured at a concentration of 1 × 106 cells/ml in 6-well plates. Inhibitors were dissolved according to the manufacturers’ guidelines and used at 1.25 µM for vorinostat (Selleckchem, Munich, Germany), 0.3 µM for mithramycin A (Sigma, St. Louis, MO, USA), 5 µM for RG108 and 5 µM 5-azacytidine (Selleckchem) for 24 h if not otherwise stated.
7
Mass Spectrometry Proteomics Protocol
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Acetonitrile (LC-MS grade) and water (LC-MS grade) were purchased from Merck (EMD Millipore). Dithiothreitol (DTT) and io-doacetamide (IAA) were purchased from Sigma-Aldrich. Formic acid (FA) was obtained from Fluka. Sequencing grade modified trypsin (proteomic grade) was acquired from Thermo Scientific. The MCF-7 cell line was obtained from American Type Cell Collection (MD, USA). Dulbecco's Modified Eagle's Medium (DMEM) was purchased from Sigma Aldrich. Trypsin-EDTA (0.05%), penicillin/streptomycin and FBS were obtained from Biochrome.
8
Cell Culture and Delivery Protocols
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HEK293T and mouse neuroblastoma N2a cells were cultured in DMEM (Life Technologies) supplemented with 10% fetal bovine serum (BIOCHROME) and 1% penicillin/streptomycin. H9 human ES cells were cultured in Essential 8 medium (Thermo, A1517001) and maintained on Matrigel‐coated six‐well plates (Corning, 354277). Primary neural progenitor cells were isolated from E13 cerebral cortices and seeded onto six‐well or 24‐well plates coated with 10 µg mL−1 poly‐d ‐lysine (Sigma‐Aldrich) and 10 µg mL−1 laminin (Invitrogen). Primary NPCs were cultured in 50% DMEM/F12 (Invitrogen) and 50% neurobasal‐A medium (Invitrogen) supplemented with 1% penicillin‐streptomycin (Invitrogen), 1× GlutaMAX (Invitrogen), 1× nonessential amino acids (Invitrogen), 2% B27 supplement (without VA) (Invitrogen), 5 ng mL−1 basic fibroblast growth factor (Invitrogen), and 2.5 ng mL−1 epidermal growth factor (Invitrogen). The differentiation medium was consisted of low‐glucose DMEM (Gibco) with 1% penicillin‐streptomycin and 2% B27 supplement (Invitrogen). Intracellular delivery of CMA and cGAMP was performed using Lipofectamine MessengerMAX. The other reagents are listed in Table S1 (Supporting Information).
9
MCF-7 Breast Cancer Cell Culture
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Human breast ductal adenocarcinoma cell line [MCF-7 (ER + /PR + )] was purchased from ATCC (American Type Culture Collection, Gaithersburg, Maryland, USA). These cells were cultured in flasks (at 37 °C and under 5% CO2 atmosphere) using RPMI medium (Biochrome, Germany) containing 10% FBS (Fetal Bovine Serum) (Biochrome, Germany) and 1% penicillin/streptomycin (100 U/mL penicillin and 100 μg/mL streptomycin) (Biochrome, Germany). After cells filled out 80% of the culture flasks, they were then washed out with 1xPBS (Gibco, USA) and detached with treatment of 0.25% of Trypsin-EDTA (Biochrome, Germany) enzymatic solution. Required amount of detached cells were used for IC50 studies, while the rest was treated with 10% DMSO in RPMI medium and stored at -80 °C for later use.
10
Epigenetic Regulation of MCC Cell Lines
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The MCC cell lines WaGa, MKL-1, MKL-2, BroLi, AlDo, LoKe54 (link)55 (link) and melanoma cell lines FM79, FM82, IF656 (link) have been described before. All cell lines were maintained in RPMI-1640 (PAN Biotech, Aidenbach, Germany) supplemented with 10% fetal bovine serum (Sigma, St. Louis, MO, USA) and 1% penicillin/streptomycin (Biochrome, Berlin, Germany). For the cell line AlDo the medium was additionally supplemented with 30% fibroblast conditioned medium. For treatment with specific inhibitors, cells were cultured at a concentration of 1 × 106 cells/ml in 6 well plates. Inhibitors were dissolved according to the manufacturers’ guidelines and used at 1.25 μM vorinostat (Selleckchem, Munich, Germany), 0.3 μM mithramycin A (Sigma) and 0.3 μM trichostatin A (Selleckchem) for 24 hours if not otherwise stated.
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