For primary sphere formation, 100 cells per 100μl stem cell media (SCM) [DMEM:F12, 1X AA, 1X B27 supplement (Cat # 17504, Gibco), 20 ng/ml epidermal growth factors (Cat # E9644, Sigma-Aldrich), and 10 ng/ml fibroblast growth factor (Cat # 354060, BD Bioscience)] were plated in non-treated, low adhesion, 96 wells plate. Four hours after incubation, vehicle or drug/metabolite at desired concentrations (as described in the figure legend) were added to each well (at least in triplicates for each sample). On day 5, numbers of spheres ranging from 50-150 micrometer in diameter were counted using a phase contrast microscope and percent inhibition was calculated compared to control.
Epidermal growth factors
Manufactured by Merck Group
Sourced in United States
Epidermal growth factors are a class of regulatory proteins that play a crucial role in cell growth, proliferation, and differentiation. They bind to specific receptors on the cell surface, triggering signaling cascades that promote cellular processes essential for tissue development and repair.
Automatically generated - may contain errors
Lab products found in correlation
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Fibroblast growth factor (fgf), by BD (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (1 mentions)
Fibroblast growth factor (fgf), by Merck Group (1 mentions)
Dmem f12 aa, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by ExCell Bio (1 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions)
4 protocols using epidermal growth factors
1
Sphere Formation Inhibition Assay
2
Cinnamomum burmannii Essential Oil Evaluation
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Cinnamomum burmannii essential oil (BEO) was provided by huaqingyuan Biotechnology Co., Ltd. (Meizhou, China). Other materials used were Dulbecco’s modified Eagle medium (Thermo fisher, Waltham, MA, USA), penicillin/streptomycin (Gibco, Waltham, MA, USA), and fetal bovine serum (EallBio, Beijing, China). Monoclonal antibodies against β-actin, and NF-κB p65 were purchased from MedChemExpress Inc. (MCE, Monmouth Junction, NJ, USA). Epidermal growth factors (Sigma–Aldrich, St. Louis, MO, USA), RNA isolater Total RNA Extraction (Vazyme, Nanjing, China), lipopolysaccharide (LPS) (Escherichia coli, serotype 0111:B4) and all other chemicals were obtained from Sigma Chemicals (St Louis, MO, USA).
3
Sphere Formation Assay for GAG Effects
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For primary sphere formation, cells were plated in non-treated, low adhesion, 96 wells plate at the concentration of 100 cells/100 μL/well in stem cell media (SCM) that consisted of DMEM:F12:AA (Gibco # 11320-023, Gibco # 15240-0062), supplemented with 1±B27 (Gibco # 17504-044), 20 ng/mL epidermal growth factors (Sigma # E9644) and 10 ng/mL fibroblast growth factor (Sigma # 354060). After four hour of incubation, vehicle (control) or GAG at the desired concentrations were added to each well (at least in triplicates for each sample). On day five, numbers of spheres ranging from 50 – 150 mm in diameter were counted using phase contrast microscope and percent inhibition was calculated compared to control.
4
Isolation and Culture of Primary Tubular Cells
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For preparing the primary cultural tubular cells (PTCs), mouse kidney tissues were filtered through the 150-µm mesh and digested with 2 mg/ml collagenase I for 30 min at 37oC with gentle stirring. Cells were harvested and cultured in Dulbecco’s modified Eagle’s medium-F12 (DMEM-F12) (cat: 12400024, Gibco, Grand Island, NY) supplemented with 10% FBS (cat: FND500, ExCell Bio, Shanghai, China), 0.5 x Insulin-Transferrin-Selenium (Glibco, Grand Island, NY), 36 ng/ml Hydrocortisone, 4 pg/ml Triiodothyronine, 10 ng/ml Epidermal Growth Factors (Sigma-Aldrich, St. Louis, MO), and 1% penicillin-streptomycin at 5% CO2, 37 oC to 80% confluence, and were digested with trypsin and planted. The primary cultured tubular cells with floxed target gene were infected with Ad-Cre to ablate target gene. Small interfering RNAs specific for mouse Zdhhc9 and APT1, respectively, were ordered from Shanghai Integrated Biotech Solutions Co., Ltd. pCMV6-DDK-tagged β-catenin WT and its mutant plasmids, pCMV6-DDK-APT1, pCMV6-EGFP-β-catenin WT and its C300S mutant (Miaolingbio, Wuhan, China) were transfected into PTCs or HEK293A cells using Lipofectamine 3000 reagent (Invitrogen, Grand Island, NY). Cells were harvested and analyzed at 36 h after transfection.
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