MC3T3-E1 cells were purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, and cultured in alpha-modified minimum essential medium eagle (α-MEM, Shanghai BasalMedia Technologies Co., LTD., China) containing 10% fetal bovine serum (FBS, Gibco, United States) and 1% penicillin-streptomycin (Gibco, United States), incubated at 37°C and 5% CO2. The medium was changed every 2 days. MC3T3-E1 was modeled by the method of serum starvation as a control group for subsequent experiments. Firstly different concentrations of BGSSD medicated serum were used to intervene MC3T3-E1 to find the optimal medicated serum concentration. Then the further experiment was performed. The Control, Epidermal growth factor (EGF), and BGSSD groups were treated for 48 h, 72 h, or 7 days with blank serum, EGF (10 ng/ml, Proteintech, United States), and optimal concentration of BGSSD containing serum, respectively.
Epidermal growth factor (egf)
Manufactured by Proteintech
Sourced in China, United States
EGF is a recombinant epidermal growth factor (rEGF) protein produced by Proteintech. EGF is a small polypeptide that stimulates cell growth, proliferation, and differentiation.
Automatically generated - may contain errors
Lab products found in correlation
Basic fibroblast growth factor (bfgf), by Proteintech (4 mentions)
R spondin, by Proteintech (2 mentions)
Noggin, by Proteintech (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Mcdb 131, by Merck Group (2 mentions)
Vascular endothelial growth factor (vegf), by Proteintech (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Lonsera (2 mentions)
24 well ultra low attachment plate, by Corning (1 mentions)
B27 supplement, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Penicillin and streptomycin, by Merck Group (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Geltrex matrix, by Thermo Fisher Scientific (1 mentions)
Fibroblast growth factor 2 (fgf2), by Proteintech (1 mentions)
Insulin, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Organoid growth medium, by STEMCELL (1 mentions)
12 protocols using epidermal growth factor (egf)
1
Optimizing MC3T3-E1 Cell Culture Conditions
2
Sphere Formation Assay for Stem Cells
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The sphere formation assay was performed in 24-well ultralow-attachment plates (#3473; Corning). LLCs (1000 cells/well) were seeded in serum-free DMEM, containing 10 mM HEPES, 10 ng/ml of basic fibroblast growth factor (#450-33-10 μg; Proteintech,), 2% B27 (serum-free supplement, #17504044; Gibco), and 20 ng/ml of epidermal growth factor (#315-09-100 μg; Proteintech). Each well was examined under a light microscope, and the total number of spheroids was counted.
3
Isolation of Breast Cancer Stem Cells
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MCF7-CSCs came from the human breast cancer cell line MCF7 that was induced as previously described (38 (link)). Serum-free medium (SFM) consisted of DMEM/F12, 20 ng/mL basic fibroblast growth factor (Proteintech, Wuhan, China), 10 ng/mL epidermal growth factor (Proteintech), and 1×B27 supplement (Gibco Life Technologies, Lofer, Austria). When MCF7 cell density reached 90%, the cell precipitate was rinsed with phosphate-buffered saline (PBS), centrifuged at 1,000 rpm for 5 minutes, and the supernatant was discarded. MCF7 cells were resuspended in SFM, and the cell suspension was transferred to non-adherent culture flasks.
4
Rat Hippocampal Neural Stem Cell Culture
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Rat NSCs were derived and cultured as described previously by others (Rietze and Reynolds, 2006 (link)). Briefly, the hippocampi of several postnatal rats were chopped, mechanical digested by 0.25% trypsin (Gibco) in a humidified 5% CO2 incubator at 37°C for 10 min and triturated. The cell suspension was added into an equal volume of DMEM/F12 (Gibco) supplemented with 10%fetal bovine serum (Lonsera) and 0.1 mg/ml DNase I (Sigma), afterward filtered through a 70 μm microfiltration membrane and centrifuged for 5 min. The cells cultured in DMEM/F12 containing 10 ng/mL basic fibroblast growth factor (Proteintech), 20 ng/mL EGF (Proteintech), 1%penicillin and streptomycin (Sigma) and 2% B27 (Gibco) without vitamin A were seeded in 6 well plate in a humidified 5% CO2 incubator at 37°C. Within 3–5 days, the cells grew into free floating neurospheres which were then gathered by centrifugation and passaged after mechanical, dissociation by pipetting.
5
Generating Mouse Intestinal Organoids
6
Culturing and Quantifying Tumor Spheres
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A total of 5 × 103 cells were seeded in low-adhesion plates in serum-free culture medium, which contained 2% B27 (Invitrogen, Carlsbad, CA, USA), EGF (Proteintech, 20 ng/ml), FGF2 (Proteintech, 10 ng/ml), insulin (Sigma, St. Louis, MO, USA; 5 μg/ml), and 0.4% bovine serum albumin (Sigma). After 2 weeks, tumor spheres with a diameter >75 μm were counted.
7
HMEC-1 Cells: Glucose and Inhibitor Exposure
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Human microvascular endothelial cell-1 (HMEC-1) cells were provided by the U533 Institute of the French National Institute of Health Medicine (Paris, French) and were cultured in MCDB131 (Sigma, St. Louis, MO, USA) containing 5 mM glucose, 10% FBS (Lonsera, Shuangru Biotech, Shanghai, China), 10 ng/mL EGF (Proteintech, Inc., Wuhan, China), and 100 U/L penicillin and 100 µg/mL streptomycin (Gibco, Carlsbad, CA, USA). The cells were cultured at 37 °C in a humidified incubator of 5% CO2 and 95% air. After reaching 80% confluence, HMEC-1 cells were exposed to HG (30 mM glucose) or mannitol (30 mM) for 48 h after pretreatment with VAC 5 µM for 12 h or other inhibitors (Compound C with 5 µM, VPA with 1 mM) and activators (AICAR with 1 mM) for 1 h.
8
Organoid Culture and DVF Treatment
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The organoid culture was performed in accordance to the protocol described previously [21 ]. In brief, organoids were generated from isolated crypts of the colon of colitis mice (C57/BL6 mice and Lrrc19−/− mice) and then embedded into Matrigel (Corning, Corning, New York, USA). After that, organoids were kept in Organoid Growth Medium (STEMCELL Technologies) in the presence of R-Spondin, Noggin, and EGF (Proteintech). To investigate the effect of DVF on organoids, organoids were co-cultured with 1 μg DVF or PBS on 6-well plates. After 5 days of co-culture, organoid morphologies were recorded and then harvested for further experiments.
9
HMEC-1 Cells Cultured and Treated
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Human microvascular endothelial cells (HMEC-1) were provided by the U533 Institute of the French National Institute of Health Medicine and were cultured in MCDB131 (Sigma St. Louis, MO, USA) containing 5 mM glucose, 10% FBS (Lonsera, Shuangru Biotech, Shanghai, China), 10 ng/mL EGF (Proteintech, Inc, Wuhan, China), and 100 U/L penicillin and 100 μg/mL streptomycin (Gibco, Carlsbad, CA, USA). The cells were incubated at 37 °C under an environment with 5% CO2. When grown to a logarithmic growth phase, HMEC-1 cells were seeded into proper plates and exposed to HG (30 mM glucose) or mannitol (30 mM) for 24 h and then treated with vaccarin (2 μM) for 12 h.
10
Protein Expression Analysis of Regenerated Epidermis
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The regenerated epidermis extracts were separated on SDS-polyacrylamide gels, and then transferred onto NC membranes (Pall, USA). The following primary antibodies were used: VEGF (1:500, proteintech, China), EGF (1:500, proteintech, China), GAPDH (1:2000, CST, USA), p-AKT (1:5000, Abcam, UK), AKT (1:5000, Abcam, UK), p-ERK (1:4000, proteintech, China), ERK (1:3000, proteintech, China), p-STAT3 (1:2000, Santa, USA), STAT3 (1:2000, Santa, USA), and GAPDH (1:2000, CST, USA).
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