To avoid any bias due to culture conditions, all cell lines were cultured in RPMI1640 medium with stable glutamine (Biochrom, Berlin, Germany), supplemented with 10% FCS tested to be dox-free (Sigma-Aldrich, Taufkirchen, Germany), and penicillin and streptomycin (final concentrations 100 units/mL and 100μg/mL, respectively; Merck, Darmstadt, Germany) in tissue culture flasks and plates (TPP, Trasadingen, Switzerland). To improve cell attachment for CHLA-10, CHLA-25, EW-3, EW-24, MIC, and TC-106, culture dishes were coated with 2% gelatine (Sigma-Aldrich) for cell expansion, and collagen type I solution from bovine skin (Sigma-Aldrich) in experimental assays. Cells were incubated at 37°C and 5% CO2 in a fully humidified environment. Cells were subcultured in ratios 1:2 to 1:8 after detachment with trypsin/EDTA (Biochrom), and spinning down the detached cells. Mycoplasma contamination was ruled out regularly by nested PCR with cell supernatant of each experiment.
Rpmi 1640 medium with stable glutamine
Manufactured by Harvard Bioscience
Sourced in Germany
RPMI 1640 medium with stable glutamine is a commonly used cell culture medium that provides essential nutrients for the growth and maintenance of various cell types. It contains a stable form of glutamine, which is a critical amino acid required for cellular metabolism and proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Streptomycin, by Merck Group (3 mentions)
Penicillin, by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Harvard Bioscience (3 mentions)
Collagen type 1 solution from bovine skin, by Merck Group (2 mentions)
Trypsin edta, by Harvard Bioscience (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Rd es, by American Type Culture Collection (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
Hek293t, by American Type Culture Collection (1 mentions)
B16 ova, by American Type Culture Collection (1 mentions)
Concanavalin a (cona), by Merck Group (1 mentions)
8 protocols using rpmi 1640 medium with stable glutamine
1
Standard Cell Culture Conditions
2
Isolation and Characterization of Peritoneal Macrophages
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Peritoneal macrophages were isolated from C57/BL6 mice. For this purpose, 2.5 ml of 4% (w/v) of Brewer's thioglycollate broth was injected into the peritoneal cavity as described previously by Schleicher et al.62 (link) Four days after the injection, the peritoneum was washed with 10 ml PBS (Gibco) and the macrophages were cultured in RPMI 1640 medium with stable glutamine (Biochrom), supplemented with 10% heat-inactivated fetal bovine serum (Biochrom), 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco) and characterized by flow cytometry with preceding F4/80 staining.
3
Establishment of Doxorubicin-resistant Ewing Sarcoma Cell Lines
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Human cell lines were provided by the following repositories and/or sources: A673, HEK293T, and RDES cells were purchased from ATCC. SK-N-MC was provided by the German Collection of Microorganism and Cell Cultures (DSMZ). TC71 and TC32 cells were kindly provided by the Children’s Oncology Group (COG). A673/TR/shEF1 cells were described previously30 (link). EW1 cells were kindly provided by O. Delattre (Institut Curie Research Center, Paris, France). All cell lines were cultured in RPMI-1640 medium with stable glutamine (Biochrom) supplemented with 10% tetracycline-free fetal calf serum (FCS; Sigma-Aldrich) and 100 U/ml penicillin and 100 µg/ml streptomycin (Merck) at 37 °C with 5% CO2 in a humidified atmosphere. Cell lines were routinely tested for mycoplasma contamination by nested PCR, and cell line identity was regularly verified by STR-profiling. Doxorubicin-resistant (Doxo-res) EwS cells were established through continuous culture with serially increasing Doxo concentrations starting at ~10 nM corresponding to pre-determined IC50 values. After successful adaptation to the given concentrations indicated by re-growth, cells were cultured with serially ascending Doxo concentrations by multiplying the IC50 values by factor 1.1–2.0. Doxo-res variants were maintained with ~200 nM Doxo.
4
Standard Cell Culture Conditions
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To avoid any bias due to culture conditions, all cell lines were cultured in RPMI1640 medium with stable glutamine (Biochrom, Berlin, Germany), supplemented with 10% FCS tested to be dox-free (Sigma-Aldrich, Taufkirchen, Germany), and penicillin and streptomycin (final concentrations 100 units/mL and 100μg/mL, respectively; Merck, Darmstadt, Germany) in tissue culture flasks and plates (TPP, Trasadingen, Switzerland). To improve cell attachment for CHLA-10, CHLA-25, EW-3, EW-24, MIC, and TC-106, culture dishes were coated with 2% gelatine (Sigma-Aldrich) for cell expansion, and collagen type I solution from bovine skin (Sigma-Aldrich) in experimental assays. Cells were incubated at 37°C and 5% CO2 in a fully humidified environment. Cells were subcultured in ratios 1:2 to 1:8 after detachment with trypsin/EDTA (Biochrom), and spinning down the detached cells. Mycoplasma contamination was ruled out regularly by nested PCR with cell supernatant of each experiment.
5
Mouse Melanoma Cell Culture
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The mouse melanoma cell lines B16-F10 and B16-OVA (ATCC, Manassas, VA, USA) both derived from C57/BL6 mice were cultured in RPMI 1640 medium with stable glutamine (Biochrom, Berlin, Germany), supplemented with 10% heat-inactivated fetal bovine serum (Biochrom), 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco, Carlsbad, CA, USA). The cells were tested negatively for mycoplasma contamination and maintained in 5% CO2 atmosphere at 37 °C and 95% relative humidity. The cells were used when they reached 90% confluence.
6
Glucocorticoid Effects on PBMC Activation
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PBMCs were treated ex vivo with DEX according to Russcher et al. [47 (link)] with modifications. Cells were thawed and resuspended in RPMI 1640 medium with stable glutamine (Biochrom, Berlin, Germany) supplemented with 10% FBS, 100 U ml−1 penicillin and 100 µg ml−1 streptomycin (all PAA). PBMCs were incubated in a shaking water bath for 30 min at 37°C to remove endogenous cortisol. The medium was replaced, and 3 × 106 cells per well were precultured overnight (5% CO2, 37°C) in a 24-well plate at a density of 6 × 106 cells ml−1. The next day, PBMCs were incubated for 4 h with increasing DEX concentrations (0, 0.5, 5, 50 and 500 nM) together with 5 µg ml−1 of the T-cell specific mitogen ConA (Sigma-Aldrich).
7
CaCo-2 Cell Culture Protocol
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CaCo-2 cells were obtained from DSMZ (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany), cultivated in 80 cm2 culture flasks in a humidified incubator at 37°C and 7% CO2 and passaged 1:6 every 3–4 days. RPMI 1640 medium (with stable glutamine; Biochrom AG) was supplemented with 10% fetal bovine serum (Biochrom AG).
8
Cultivation and Supernatant Preparation of B. thuringiensis
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The B. thuringiensis strains used in this study are listed in Table 1 . For gaining cell-free culture supernatants, they were pre-cultured in casein glucose yeast (CGY) medium with 1% glucose for 17 h at 32 °C and 200 rpm. Samples of 20 mL fresh CGY medium were inoculated to an optical density (OD600) = 0.2 and cultured for further 6 h. After centrifugation at 3500 rpm and 4 °C for 12 min, 1 mM ethylenediaminetetraacetic acid (EDTA) was added, the supernatants were filtered through a 0.2 µm filter, aliquoted and stored at −20 °C. For cultivation under simulated intestinal conditions, preparation of “conditioned” (cRPMI) medium was carried out as described previously [15 (link)]. In brief, RPMI 1640 medium (with stable glutamine; Biochrom AG, Berlin, Germany), supplemented with 1% glucose and 2% casein hydrolysate, was incubated with differentiated CaCo-2 cells for 22 h and filtered using a 0.2 µm filter. Overnight cultures obtained as described above were incubated at 37 °C and adjusted to a start OD600 of 0.05 in 20 mL cRPMI. They were incubated statically at 37 °C and 7% CO2 atmosphere. For growth analyses, OD600 was measured every hour for eight hours. Cell-free culture supernatants were harvested after six hours as described above.
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