SH-SY5Y, a human-derived neuroblastoma cell line, is thrice-cloned originally from SK-N-SH and widely used in the scientific research of neurodegenerative disorders.32 (link) SH-SY5Y was grown in Dulbecco’s Modified Eagle’s Medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA) and 1% penicillin/streptomycin. Cells were maintained in a humidified atmosphere at 37°C with 5% CO2. The autophagy inhibitor (3-methyladenine, 3-MA, Santa Cruz Biotechnology, Dallas, TX, USA) and inducer (STF-62247, Selleck Chemicals, Houston, TX, USA) were dissolved in dimethyl sulfoxide and used at the following concentrations: the inhibitor (3-MA), 10 mM; the inducer (STF-62247), 10 μM. The cells were treated with the autophagy inhibitor and inducer without fetal bovine serum for 24 hours.
Fetal bovine serum (fbs)
Manufactured by Selleck Chemicals
Sourced in United States
Fetal bovine serum (FBS) is a widely used cell culture supplement derived from the blood of bovine fetuses. It provides essential nutrients, growth factors, and other components that support the growth and proliferation of cells in vitro. FBS is a complex mixture that is commonly used in cell culture media to promote the survival and expansion of various cell types.
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Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Enzalutamide, by Selleck Chemicals (2 mentions)
Fetal bovine serum (fbs), by Corning (2 mentions)
Dulbecco modified eagle medium (dmem), by Selleck Chemicals (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
3 methyladenine 3 ma, by Santa Cruz Biotechnology (1 mentions)
Stf 62247, by Selleck Chemicals (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Oleic acid, by Selleck Chemicals (1 mentions)
Oleic acid, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Cytiva (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Minimum essential medium (mem), by GE Healthcare (1 mentions)
Imdm medium, by Thermo Fisher Scientific (1 mentions)
Dmem medium, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Kyse30, by Procell (1 mentions)
Puromycin, by Selleck Chemicals (1 mentions)
Kyse150, by Procell (1 mentions)
8 protocols using fetal bovine serum (fbs)
1
Modulating Autophagy in SH-SY5Y Cells
2
Intracellular Lipid Quantification in Rat Hepatoma Cells
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McArdle rat hepatoma cells (McA-RH 7777; LGC Standards AB, Borås, Sweden) were cultured in MEM (PAA Laboratories GmbH, Linz, Austria) containing 20% fetal bovine serum (HyClone Laboratories, South Logan, UT, USA). For assessment of intracellular lipids, the cells were plated in 24-well plates containing glass coverslips and grown in MEM with 2% fetal bovine serum. After 24 hours, the medium was changed to MEM without fetal bovine serum plus 0, 20, or 60 µM oleic acid (Sigma-Aldrich), and after 48 hours, the cells were treated with 1 nM DHT (dissolved in ethanol; Sigma-Aldrich) or vehicle for 24 hours. In separate experiments, the medium was changed to MEM without fetal bovine serum plus 60 µM oleic acid 24 hours after plating, and after 48 hours, the cells were treated with 1 nM DHT, 10 μM enzalutamide (Selleckchem, Houston, TX, USA), or vehicle for 24 hours. Cells were then fixed in 2% formaldehyde for 5 minutes, treated with 20% isopropanol for 0.5 minutes, and stained with Oil Red O for 20 minutes. Next, cells were treated with 20% isopropanol for 0.5 minutes, stained with hematoxylin for 2 minutes, and finally rinsed in tap and distilled water. Cells were photographed with a Zeiss microscope and Oil Red O–stained area per cell was quantified by BioPix iQ 2.1.8 software (BioPix Software).
3
Culturing Prostate Cancer Cell Lines
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LNCaP, C4–2, 22RV1, and PC-3 cells were obtained from ATCC and cultured in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (Corning) and 1% penicillin-streptomycin (p/s) (Gibco). CS2 cells were cultured in RPMI 1640 medium (Gibco) supplemented with 10% charcoal-stripped serum (Corning) and 1% p/s (Gibco). VCaP was cultured in DMEM medium (Thermo) supplemented with 10% fetal bovine serum (Corning) and 1% p/s (Gibco) and LAPC4 was cultured in IMDM medium (Thermo) supplemented with 10% fetal bovine serum (Corning) and 1% p/s (Gibco). For androgen ablation experiments, LNCaP cells were cultured in RPMI medium containing 10% charcoal-stripped serum +1% p/s for 7 and 14 days, respectively prior to harvesting for flow cytometry. For AR inhibition experiments, LNCaP cells were cultured in RPMI medium containing 10% fetal bovine serum + 1% p/s + 10 µM Enzalutamide (Selleckchem). LNCaP cells to be studied as a control for the Enzalutamide-treated group were cultured in RPMI medium containing 10% fetal bovine serum + 1% p/s + DMSO (equivalent amount). All cell lines were authenticated prior to use.
4
Esophageal Squamous Cell Carcinoma Cell Line Cultivation
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Four ESCC cell lines (KYSE140, KYSE180, KYSE450, and KYSE510) and one immortalized esophageal cell line (NE1) were kindly provided by Professor Li Fu (Department of Pharmacology and International Cancer Center, Shenzhen University). The other four ESCC cell lines KYSE30, KYSE150, KYSE410, and TE-1 were purchased from Procell (Procell Life Science&Technology Co,. Ltd, Wuhan, China). The ESCC cell lines were cultured in RPMI-1640 (Gibco, USA) supplemented with fetal bovine serum (Gibco, USA) and 1% penicillin/streptomycin (Gibco, USA). The stable overexpression and knockdown cell lines KYSE30, KYSE410, KYSE510, and TE-1 were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 1 μg/mL puromycin (Selleck Chemicals, USA). NE-1 was cultured in keratinocyte serum-free medium (K-SFM) and EpiLife at a ratio of 1:1 supplemented with 0.05 mg/mL bovine pituitary extract (BPE) and 5 ng/mL human recombinant epidermal growth factor (EGF). For verteporfin treatment, cells were incubated with 2.5 μM (MCE, USA) for KYSE30 in FBS-free RPMI-1640 containing 0.1% BSA for 6 h before cells were harvested. Cells were cultured at 37°C in a humidified atmosphere of 5% CO2. The cell lines were authenticated by short tandem repeat profiling (STR) (Biowing Applied Biotechnology Co. Ltd, Shanghai, China) and to be free of mycoplasma contamination.
5
Regulation of Kidney Cell Signaling
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Human kidney proximal epithelial cells (HK-2) and conditionally immortalized mouse podocytes were purchased from the China Center for Type Culture Collection (CCTCC). HK-2 was cultured in DMEM-F12 supplemented with 10% fetal bovine serum (GIBCO, Carlsbad, CA, USA), and grown at 37 C with 5% CO 2 . MPC5 was cultured in DMEM containing 5.5 mM glucose with 10% fetal bovine serum and grown at 33 C with 5% CO2 and differentiated at 37 C. HK-2 and podocytes were stimulated with Ang II at doses of 0, 0.01, 0.1, 0.5, 1.0, 2.0 mM and 1.0 mM for 0, 1, 3, 6, 12, 24 and 48 h.
Angiotensin II type 1 receptor blocker losartan (LOS, 1 mM, Selleck Chemicals, Huston, USA) and Wnt/b-catenin inhibitor ICG-001 (ICG, 10 mM; Selleck Chemicals, Huston, USA) were used. The Wnt/b-catenin inhibitor ICG-001 inhibits b-cateninemediated gene transcription. To discover novel inhibitors of RAS and Wnt/bcatenin signaling pathway and anti-fibrotic candidate drugs, ERG, ABA and PAB were isolated from natural products Polyporus umbellatus [31] , Alisma orientale [32] and surface layer of Poria cocos [33] . In some experiments, cells were incubated with ERG, ABA and PAB at 10 mM. We further investigate the inhibition effects of theses natural products on RAS and Wnt/b-catenin signaling pathways. Whole-cell lysates were prepared and subjected to Western blot analysis, and total RNA was extracted for PCR analysis.
Angiotensin II type 1 receptor blocker losartan (LOS, 1 mM, Selleck Chemicals, Huston, USA) and Wnt/b-catenin inhibitor ICG-001 (ICG, 10 mM; Selleck Chemicals, Huston, USA) were used. The Wnt/b-catenin inhibitor ICG-001 inhibits b-cateninemediated gene transcription. To discover novel inhibitors of RAS and Wnt/bcatenin signaling pathway and anti-fibrotic candidate drugs, ERG, ABA and PAB were isolated from natural products Polyporus umbellatus [31] , Alisma orientale [32] and surface layer of Poria cocos [33] . In some experiments, cells were incubated with ERG, ABA and PAB at 10 mM. We further investigate the inhibition effects of theses natural products on RAS and Wnt/b-catenin signaling pathways. Whole-cell lysates were prepared and subjected to Western blot analysis, and total RNA was extracted for PCR analysis.
6
Kidney Explant Culture Assay
7
Establishing Mouse Embryonic Stem Cell Lines
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Diploid or tetraploid blastocysts 4.5 dpc were transferred to a gelatin (Millipore)-coated cell culture dish with mitomycin-C-treated mouse embryonic fibroblast (MEF) feeder cells and cultured in stem cell medium comprising DMEM (Gibco) with 20% FBS (Gibco), 1% NEAA (Gibco), 1% GlutaMAX-L (Gibco) and 1% penicillin/streptomycin (Gibco); 0.1 mM b-mercaptoethanol; and 1,000 units/ml ESGRO leukemia inhibitory factor (LIF; Millipore). After 6–7 days, the colonies were digested with TrypLE (Invitrogen), transferred to a new gelatin-coated cell culture dish with mitomycin C-treated MEFs, and cultured in fresh medium; these cells were designated as P1. Then, the ESCs from P2 were cultured in stem cell medium comprising DMEM with 15% FBS, 1% NEAA, 1% GlutaMAX-L, 1% penicillin/streptomycin, 0.1 mM b-mercaptoethanol, 1 μM PD0325901 (Selleck), 3 μM CHIR99021 (Selleck) and 1,000 units/ml LIF.
8
Endothelial Cell Culture and Inhibitor Treatment
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Human umbilical vein vascular endothelial cells isolated from human umbilical vein vascular (Catalog Number: 8000, ScienCell, San Diego, California) were cultivated by Endothelial Cell Medium (ECM, Cat. No: 1001, ScienCell) with 5% of foetal bovine serum (FBS, Cat. No: 0025, ScienCell), 1% endothelial cell‐derived growth factor (ECGS, Cat. No: 1052, ScienCell) and 1% penicillin/streptomycin (P/S, Cat. No: 0503, ScienCell) and were incubated in 37°C and 5% CO2. LPS (LPS from Escherichia coli 055: B5, Cat. No: L2880, Sigma, Germany), simvastatin (Cat. No: S6196, Sigma, Germany), PI3K inhibitor LY294002 (#9901, Cell Signaling Technology, USA) and PDGFRβ inhibitor Imatinib (Cat. No: STI571, Selleck, USA) were attenuated by ECM without FBS, and HUVECs were interfered for 24 hours with LPS doses of 10 μg/mL, and SV/SV‐NPs were used at the concentration of 1 μmol/L, LY294002 was used at the concentration of 25 μmol/L, and Imatinib was used at the concentration of 20 μmol/L.
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