Cells were grown for 4 days in a hormone-stripped DMEM devoid of phenol red and supplemented with 3% steroid-depleted, charcoal-treated foetal calf serum (Biowest, Riverside, MO, USA); 104 cells per well were plated onto a 96-well plate in charcoal-stripped medium. The following day, the cells were treated with 4-hydroxy-tamoxifen (OH-Tam, Sigma) at different concentrations for 72 h. Cell viability was analysed using the Cell Titer 96® aqueous one solution Cell Proliferation kit from (Promega, Fitchburg, WI, USA). Briefly, the reagent (MTS) was added in each well, incubated for 1 h at 37 °C, and the absorbance was recorded at 490 nm following the manufacturer’s recommendations.
Foetal calf serum
Manufactured by Biowest
Sourced in France
Foetal calf serum is a cell culture supplement derived from the blood of bovine foetuses. It provides a complex mixture of proteins, growth factors, and other components that support the growth and maintenance of cells in vitro.
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Lab products found in correlation
4 hydroxy tamoxifen oh tam, by Merck Group (1 mentions)
Celltiter 96 aqueous one solution cell proliferation kit, by Promega (1 mentions)
Penicillin and streptomycin, by Lonza (1 mentions)
Capan 2, by American Type Culture Collection (1 mentions)
Capan 1, by American Type Culture Collection (1 mentions)
Docetaxel, by Merck Group (1 mentions)
Dulbecco s modified eagle medium, by Lonza (1 mentions)
Rpmi medium, by Lonza (1 mentions)
Dulbecco modified eagle medium (dmem), by Fujifilm (1 mentions)
Neuro 2a, by American Type Culture Collection (1 mentions)
Cell culture flasks, by Sarstedt (1 mentions)
Ham s f10, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Senescence β galactosidase staining kit, by Cell Signaling Technology (1 mentions)
Heavy 13c6 lysine and 13c6 15n4 arginine, by Cambridge Isotopes (1 mentions)
12c614n4 arginine, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin fungizone, by Thermo Fisher Scientific (1 mentions)
Facs lysing solution, by BD (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
10 protocols using foetal calf serum
1
Cell Viability Assay with Tamoxifen
2
Comparative analysis of gemcitabine-sensitive and resistant pancreatic cancer cell lines
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In this study, a gemcitabine-sensitive cell line, PANC1, and a gemcitabine-resistant PANC1 cell line, PANC1R, were kindly supplied by the Institute for Surgical Research, Philipps-University of Marburg, Marburg, Germany.13 (link) Cells were cultured in Roswell Park Memorial Institute (RPMI) medium (Lonza, Switzerland) supplemented with 10% heat-inactivated foetal calf serum (Biowest, Nuaillé, France) and 1% penicillin and streptomycin (Lonza, Switzerland). Cells were maintained at low passage and tested negatively for mycoplasma contamination. Hs766t cells and Capan-2 (ATCC, Manassas, USA) were cultured in supplemented Dulbecco’s Modified Eagle Medium (Lonza) and several primary cell lines (PDAC1-4)15 (link) and Capan-1 (ATCC) were grown at low density in supplemented RPMI medium. Paclitaxel (Bristol-Meyers Squibb, New York, USA) and docetaxel (Sigma-Aldrich, St. Lous, USA) were dissolved in 50% ethanol and sterile H2O. Gemcitabine was kindly provided by Eli Lilly Corporation (Indianapolis, USA) and dissolved in sterile water. Nab-paclitaxel was kindly supplied by the Pharmacy of New York Presbyterian Hospital (New York, NY, USA) and was dissolved in NaCl.
3
Culturing Murine Microglial and Neuronal Cells
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The murine microglial cell line BV2 and the neuronal cell line Neuro 2A were purchased from ATCC (Manassas, VA, USA). Both cell lines were maintained in DMEM (Wako, Osaka, Japan) supplemented with 10% foetal calf serum (Biowest, Nuaille, France) and antibiotics (Wako).
4
Culturing Murine Monocytic RAW264.7 Cells
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RAW264.7 cells (murine monocytic cell line; ATCC) were cultured in DMEM, supplemented with 10 % heat-inactivated foetal calf serum (Bio West) and 100 U/ml penicillin and 10 μg/ml streptomycin (PAA). The cells were cultivated in cell culture flasks (75 cm2; Sarstedt) at humidified atmosphere (95 %), 5 % CO2 and 37 °C temperature. The cells were split every 2–3 days in a ratio of 1:30.
5
Culturing and Labeling Cell Lines
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MRC5 human fibroblasts were cultured in a 1:1 ratio of Dulbecco’s modified Eagle medium and Ham’s F10 (Invitrogen) supplemented with 10% foetal calf serum (Biowest) and 1% penicillin–streptomycin (Sigma-Aldrich) at 37 °C and 5% CO2 in a humidified incubator.
TIG3 cells were grown in Dulbecco’s modified Eagle medium containing 10% FBS and 1% penicillin–streptomycin supplemented with MEM non-essential amino acid mix. Quiescent cells were obtained by contact inhibition. SA-β-galactosidase assay was performed using Senescence β-Galactosidase Staining Kit from Cell Signaling, following manufacturer instructions.
mESCs were maintained in 2i medium deficient in lysine, arginine andl -glutamine (PAA) at 37 °C and 5% CO2 in a humidified incubator. For SILAC labelling, cells were grown in a medium containing 73 µg ml−1 light [12C6]-lysine and 42 µg ml−1 [12C6, 14N4]-arginine (Sigma-Aldrich) or similar concentrations of heavy [13C6]-lysine and [13C6, 15N4]-arginine (Cambridge Isotope Laboratories).
TIG3 cells were grown in Dulbecco’s modified Eagle medium containing 10% FBS and 1% penicillin–streptomycin supplemented with MEM non-essential amino acid mix. Quiescent cells were obtained by contact inhibition. SA-β-galactosidase assay was performed using Senescence β-Galactosidase Staining Kit from Cell Signaling, following manufacturer instructions.
mESCs were maintained in 2i medium deficient in lysine, arginine and
6
Differentiation and Treatment of 3T3-L1 Adipocytes
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3T3-L1 adipocytes cell lines were cultured using Dulbecco’s modified Eagles medium (DMEM) (GIBCO, USA) supplemented with 10% Foetal calf serum (BioWest, France) and 1% penicillin/streptomycin/fungizone (GIBCO, USA) at 37 °C with 5% CO2 and 95% humidity. The maintenance of the cells was done in continuous passage by trypsinization of subconfluent cultures with Trypsin/Versene (Highveld, RSA). Differentiation was induced by introduction of medium containing 2% Foetal calf serum and 2% penicillin/ streptomycin/fungizone when pre-adipocytes were 80% confluent. Cells were kept in this medium for 5 days until adipocytes were well formed. Differentiated 3T3-L1 pre-adipocytes were treated with 5 μL of compounds (10 mg/mL) 2b-h or 100 nM insulin or Metformin for 4 h.
7
Whole Blood Stimulation and Cryopreservation
8
Stable PLOD3 Expression in HeLa Cells
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The pPuro-DHFR-LH3 construct was transfected into human cervical carcinoma cells (HeLa S3, provided by ATCC and further selected for high transfection efficiency by Dr.F. Peverali, Consiglio Nazionale delle Ricerche, Pavia) using the Lipofectamine LTX reagent (Invitrogen). Cells were not authenticated and not tested for mycoplasma contamination. Stably transfected clones, isolated with 1 mg mL−1 puromycin (Invivogen), were subjected to step-wise selection with increasing methotrexate concentrations to select for cells containing multiple copies of the plasmid. Cells were routinely cultured at 37 °C in 5% CO2 in high-glucose DMEM supplemented with 10% foetal calf serum (Biowest), 1× non-essential amino acids, 2 mM L-glutamine and 1× penicillin-streptomycin. Clones expressing high yields of PLOD3 were identified by SDS-PAGE analysis after imidazole elution from small-scale immobilized metal ion affinity purification using Nickel sepharose beads (GE Healthcare).
9
Normal Human Dermal Fibroblast Culture
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Adult normal human dermal fibroblasts (NHDF) from 37- to 50-year-old women and 45- to 57-year-old men were obtained from Genlantis (San Diego, CA, USA), Cambrex (Walkerville, MI, USA) and Coriell (Camden, NJ, USA). NHDF were maintained as monolayer cultures in 100 mm × 20 mm cell culture dishes with Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific, San Diego, CA, USA) supplemented with 10% (v/v) foetal calf serum (Biowest, Nuaillé, France), 4 mmol/L l-glutamine and 100,000 U/L penicillin, 100 mg/L streptomycin, 0.25 mg/L amphotericin B (PAN Biotech, Aidenbach, Germany) at 37 °C, 5% CO2. Cells were used in Passage 4–9, corresponding to a cumulative population doubling levels of 7 to 17. Therefore, primary NHDF were subcultured with an expansion ratio not exceeding 1:3 upon near confluence using 0.05% trypsin (PAN Biotech, Aidenbach, Germany).
10
Catalase Overexpression in RINm5F Cells
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RINm5F insulin-producing cells were cultured in RPMI 1640 medium supplemented with 10 mM glucose, 10% (v/v) foetal calf serum (Biowest, Nuaillé, France), penicillin, and streptomycin in a humidified atmosphere at 37°C and 5% CO 2 , as described previously [23] . RINm5F cell clones that overexpressed catalase either in the mitochondria or in the peroxisomes were generated as described previously [24, 25] . Cellular expression of catalase was analysed by enzyme activity measurement.
NEFAs (Sigma, St. Louis, MO, USA) were dissolved in 90% ethanol by heating to 60°C and used at different concentrations in RPMI 1640 (PAN, Aidenbach, Germany) with 1% foetal calf serum and a final BSA/NEFA ratio of 2%/1 mM (defined NEFA free BSA, MP Biomedicals, Eschwege, Germany). All untreated wells received the same amount of solvent and BSA. This procedure did not cause a significant decrease in viability in the absence of fatty acids [26] .
NEFAs (Sigma, St. Louis, MO, USA) were dissolved in 90% ethanol by heating to 60°C and used at different concentrations in RPMI 1640 (PAN, Aidenbach, Germany) with 1% foetal calf serum and a final BSA/NEFA ratio of 2%/1 mM (defined NEFA free BSA, MP Biomedicals, Eschwege, Germany). All untreated wells received the same amount of solvent and BSA. This procedure did not cause a significant decrease in viability in the absence of fatty acids [26] .
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