Human embryonal kidney (HEK) 293 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Life Technologies, Gibco, Darmstadt, Germany) supplemented with 10% fetal calf serum (FCS) and 1% of each penicillin/streptomycin (Sigma Aldrich Chemie GmbH, München, Germany). Primary neonatal rat CM were isolated from ventricular tissue of 1-3-day-old Wistar rat pups (Charles River, Sulzfeld, Germany) and cultured in CMRL 1415 medium (Biochrom AG, Berlin, Germany) supplemented with, 5.4 mM KCl, 1.26 mM CaCl2, 10 mM Hepes, 2 μM 5′-Fluoro-2′-deoxyuridine (Sigma-Aldrich), 10% FCS and 2 μg/ml of gentamycin (Biochrom AG) as described previously [11] (link), [39] (link). The seeding cell density using 6-well Nunc™ Cell-Culture dishes with surface coating Nonclon Delta (Cat-No. 140685, Thermo Fisher Scientific; Rockford, IL, USA) was 1.25×105 cells/cm2. Hearts were removed from decapitated rat pups and used for isolation of CM in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Decapitation and removal of hearts was approved by the Landesamtes für Gesundheit und Soziales (LAGeSo) in Berlin, Germany under the Permit Number: O 0139/05.
Nunc cell culture dishes
Manufactured by Thermo Fisher Scientific
Sourced in United States
Nunc™ Cell-Culture dishes are sterile, single-use, and made of high-quality polystyrene. They are designed to provide a suitable surface for the growth and maintenance of various cell lines in cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin l glutamine, by Thermo Fisher Scientific (2 mentions)
Wistar rat pups, by Charles River Laboratories (1 mentions)
Gentamycin, by Harvard Bioscience (1 mentions)
5 fluoro 2 deoxyuridine, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
High glucose dmem, by Corning (1 mentions)
Mouse m csf, by Fujifilm (1 mentions)
Amphotericin b, by Thermo Fisher Scientific (1 mentions)
M csf, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Omega Scientific (1 mentions)
Low glucose, by Thermo Fisher Scientific (1 mentions)
Pen strep glutamine, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Inverted light microscope, by Leica (1 mentions)
Laminin, by Merck Group (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Pcdna3.1 n egfp, by GenScript (1 mentions)
Egfp c 1, by GenScript (1 mentions)
6 protocols using nunc cell culture dishes
1
Isolation and Culture of Rat Cardiomyocytes
2
Generating Hamster Bone Marrow-Derived Macrophages
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Hamster bone marrow-derived macrophages (BMDMs) were generated as detailed previously in (31 (link)). Femur, tibia and iliac bones were flushed with DMEM high glucose (Corning), red blood cells were lysed, and cells cultured in DMEM high glucose (50%), 30% L929-cell conditioned laboratory-made media (as source of macrophage colony-stimulating factor (M-CSF)), 20% FBS (Omega Scientific), 100 U/ml penicillin/streptomycin+L-glutamine (Gibco) and 2.5 μg/ml Amphotericin B (HyClone). After 4 days of differentiation, 16.7 ng/ml mouse M-CSF (Shenandoah Biotechnology) was added. After an additional 2 days of culture, non-adherent cells were washed off with room temperature DMEM to obtain a homogeneous population of adherent macrophages which were seeded for experimentation in Nunc Cell Culture dishes (Thermo Scientific) overnight in DMEM containing 10% FBS, 100 U/ml penicillin/streptomycin+L-glutamine, 2.5 μg/ml Amphotericin B and 16.7 ng/ml M-CSF. For Kdo2-Lipid A (KLA), activation, macrophages were treated with 10 ng/ml KLA (Avanti Polar Lipids) for 1 hour.
3
Generation and Characterization of CIZ1-null Mouse Primary Cells
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CIZ1-null mice were generated as described [14 (link)] from C57BL/6 ES clone IST13830B6 (TIGM) harbouring a neomycin resistance gene trap inserted into intron 1. Murine primary embryonic fibroblasts (PEFs) were derived from individual embryos at days 13–14 of gestation and murine tail-tip fibroblasts (TTFs) at 3–4 weeks (Additional file 7 : Table S2). Both were maintained in Dulbecco's Modified Eagle Medium (DMEM), GlutaMAX, high glucose (4.5 g/l), and low glucose (1 g/l) (Gibco), respectively, and grown on Nunc™ Cell Culture Dishes (ThermoFisher Scientific, 150,350) with 0.13–0.16 mm thick glass coverslips. Media was supplemented with 10% FBS (PAA) and 1% Pen/Strep/Glutamine (PSG, Gibco) referred to here as high-serum media. Cells were maintained in a rapidly cycling state at 37 °C with 5% CO2 and split to avoid cell contact. All cell populations were used at passages 2–4. For inducible cells harbouring transactivator and responder transgenes, addition of doxycycline (dox) to media (5–10 μg/ml) was used to induce GFP-CIZ1 over 48 h.
4
Trypan Blue Assay for Breast Cancer Cell Viability
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Trypan blue exclusion assay was used for or a direct identification and enumeration of live and dead MDA-MB-231 and MCF-7 breast cancer cells. Cells were plated in 35mm cell culture dishes (Thermo Scientific Nunc Cell Culture Dishes; Thermo Scientific; USA) with 3 × 104/ml density and incubated overnight before the AWB extract treatment (10000, 5000, 2500, 1250, 625, 312.5 and 156.25 μg/ml for 24, 48, 72 h period). Controls received only cell culture media. Percentage of alive cells in the assay was calculated from 20 randomly selected areas [13 ] upon investigation under inverted light microscope (Leica Microsystems, Germany).
5
Culturing Mouse Neuronal Cells
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C57BL/6 wild-type and SARM1−/− mice were obtained from Babraham Institute (Babraham, UK). C57BL/6OlaHsd-Wlds (Wlds) mice, originally obtained from Harlan (Bicester, UK), were maintained as a separate colony at the Babraham Institute for 13 years. All animal work was performed in accordance with the 1986 Animals (Scientific Procedures) Act under project licence PPL 70/7620. All superior cervical ganglion explant (SCGe) and dissociated superior cervical ganglion (dSCG) cultures were obtained from humanely killed postnatal day 0 (P0) to P2 mice and were prepared as described previously 7 (link),8 (link). Cells were cultured for 7 days in vitro before experimentation. SCGe were plated on 35 mm Nunc cell culture dishes (Thermo-Fisher Scientific, Waltham, Massachusetts, USA), and dSCGs were plated on low 35 mm μ-dishes (ibidi, Martinsried, Germany). Primary cortical neuronal cells were extracted by dissecting out a section of cortical mantle, trypsinizing and dissociating the neurons before separating by centrifugation. Viability was confirmed to be greater than 95%. The cells were then plated and cultured on low 35 mm μ-dishes (ibidi) for 7 days in vitro before experimentation. In all cases, dishes were coated with poly-l -lysine (25 μg/ml; Sigma, St. Louis, Missouri, USA), then laminin (2 μg/cm2; Sigma). All cultures were maintained in an incubator at a constant 37°C with 5% CO2.
6
Stable Expression of hSERT-GFP in HEK293
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hSERT tagged with GFP was stably expressed in a tetracycline-inducible HEK293 cell line. HEK293 cells have been previously authenticated by short tandem repeat profiling at the Medical University of Graz (Cell Culture Core Facility, Graz, Austria). Cercopithecus aethiops SV40-transformed kidney (Cos-7) cells were obtained from American Type Culture Collection (CRL-1651; Manassas, VA). Cos-7 were transfected with a plasmid encoding N-terminally GFP-tagged hGlyT1b or hGlyT2a, respectively; the complementary DNAs of hGlyT1b in eGFP-C-1 and hGlyT2b in pcDNA3.1(+)-N-eGFP were bought from GenScript (New York, NY). The transfection was performed with jetPRIME (0.8 μg DNA/dish) according to the manufacturer’s protocol. All cells were maintained in Dulbecco's Modified Eagle’s Medium containing 10% fetal bovine serum. Twenty-four hours before the experiment, the cells were seeded at low density onto poly-D-lysine-coated dishes (35 mm Nunc Cell culture dishes, Thermo Fisher Scientific, Waltham, MA).
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