Immortalized skeletal myoblasts were derived from Plec−/− and wild-type littermates, both crossed into a p53−/− background, and cultivated in Ham's F10 medium, supplemented with 20% fetal calf serum, 2.5 ng/ml basic fibroblast growth factor (bFGF, Promega) and antibiotics, on collagen-coated culture dishes, as described in Reference (11 (link)). To induce differentiation, cultures were switched to Dulbecco's modified Eagle's medium containing 5% horse serum.
B fgf
Manufactured by Promega
Sourced in United States
B-FGF is a recombinant human basic Fibroblast Growth Factor (bFGF) protein. It plays a key role in the regulation of cell growth, proliferation, and differentiation.
Automatically generated - may contain errors
Lab products found in correlation
Epidermal growth factor (egf), by Promega (9 mentions)
Mcf 7, by Thermo Fisher Scientific (4 mentions)
Dmem f12, by Thermo Fisher Scientific (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Mda mb 231, by American Type Culture Collection (2 mentions)
Mcf 10a, by American Type Culture Collection (2 mentions)
Mcf 7, by American Type Culture Collection (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Ultra low attachment 96 well plate, by Corning (1 mentions)
Horse serum, by Thermo Fisher Scientific (1 mentions)
Ham s f10 nutrient mixture, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Hcc1937, by American Type Culture Collection (1 mentions)
Lipofactamine 3000, by Thermo Fisher Scientific (1 mentions)
Mda mb 231, by Thermo Fisher Scientific (1 mentions)
Hcc1937, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Puromycin, by Merck Group (1 mentions)
Heracell 150i co2 incubator, by Thermo Fisher Scientific (1 mentions)
L 685 458, by Merck Group (1 mentions)
13 protocols using b fgf
1
Immortalized Skeletal Myoblast Derivation
2
Culturing Breast Cell Lines
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Normal breast epithelial cell line MCF-10A, breast cancer cell lines MCF-7 and T47D were obtained from ATCC (Manassas, VA, USA). MCF-10A, MCF-7 and T47D cell lines were cultured in high-glucose (4.5 mg/ml) DMEM with 10% (v/v) FBS (HyClone). MCF-7 CSCs and T47D CSCs were induced and cultured in DMEM-F12 (Gibco, Thermo Fisher Scientific), containing 2% B27 (Gibco), b-FGF 10 μg/L (Promega), EGF 20 μg/L (Promega), as previously described [23 (link)]. All cells were maintained at 37 °C in a 5% CO2 and 95% air incubator.
3
Mammosphere Formation Assay
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After indicated treatment, cells were suspended in serum-free DMEM/F12 (HyClone, USA) that contains 20 ng/mL EGF (Promega, USA), 20 ng/mL b-FGF (Promega, USA), and 2% B27 (Invitrogen, USA) as single cells. The cells were then incubated in an ultra-low attachment 96-well plate (Corning, USA) at a density of 5×103 cells per well. After incubation for 10 days, mammospheres were captured under a microscope (Leica, Germany).
4
Isolation and Culture of Avian and Murine Myoblasts
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Chicken primary myoblasts were isolated from the chicken leg and breast muscle of day 10 embryo as previous described.12 Primary myoblast represented the chicken primary myoblasts that have just completed serial plating. Growing myoblast represented myoblasts that were cultured in growth medium with RPMI‐1640 (Gibco, Grand Island, NY, USA), 15% foetal bovine serum (FBS) (ExCell, Shanghai, China), 10% chicken embryo extract, and 0.2% penicillin/streptomycin (Gibco) at 37°C in 5% CO2. Differentiated myotube (DM) represented myoblasts induced to differentiation for 4 days by culturing the cells in differentiation medium (RPMI‐1640 without FBS containing 2% horse serum) when 90% confluent.
Mouse primary myoblasts were isolated and cultured as previously described.13 Cells were isolated from the forelimbs and hindlimbs of 3‐week‐old mice, minced and digested in a solution of dispase B and type I collagenase. Growth medium consisted of Ham's F‐10 nutrient mixture (Gibco) supplemented with 20% FBS (ExCell) and 2.5 ng/mL bFGF (Promega, Madison, WI, USA). Differentiation medium consisted of DMEM (Gibco) supplemented with 2% horse serum (Gibco). All medium contained 0.2% penicillin/streptomycin (Gibco).
Mouse primary myoblasts were isolated and cultured as previously described.
5
Isolating and Culturing Primary Myoblasts
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Primary myoblasts were isolated from neonatal mice as previously described (37 ). Primary myoblasts were further enriched by preplating 30 min for each passage until near 100% cells were positive for desmin. Primary myoblasts were maintained in Ham’s F-10 nutrient mixture-based growth medium containing 20% fetal bovine serum (FBS), 2.5% chicken embryo extract (CEE) (US Biologicals), 5 ng/mL basic fibroblast growth factor (bFGF) (Promega) and 100 U/mL penicillin and 100 μg/mL streptomycin. Differentiation medium DMEM (Dulbecco's modified Eagle medium) with 2% horse serum) was used to induce primary myoblast differentiation.
Single myofibers were isolated from the EDL muscle as previously described (38 (link)). For immediate quantification of satellite cells, single fibers were fixed in 4% paraformaldehyde for 10 min at room temperature. Fibers were permeabilized with PBS-T (phosphate-buffered saline [PBS] with 0.5% Triton X-100) for 15 min and blocked with blocking solution (2% bovine serum albumin [BSA]/5% goat serum/0.1% Triton X-100 in PBS) for 1 h at room temperature.
Single myofibers were isolated from the EDL muscle as previously described (38 (link)). For immediate quantification of satellite cells, single fibers were fixed in 4% paraformaldehyde for 10 min at room temperature. Fibers were permeabilized with PBS-T (phosphate-buffered saline [PBS] with 0.5% Triton X-100) for 15 min and blocked with blocking solution (2% bovine serum albumin [BSA]/5% goat serum/0.1% Triton X-100 in PBS) for 1 h at room temperature.
6
Modulating Breast Cancer Stemness
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Triple-negative breast cancer cell lines MDA-MB-231 and HCC-1937 were obtained from ATCC (Manassas, VA, USA). Cell lines were cultured in DMEM (Gibco) with 10% FBS (Gibco). MDA-MB-231 CSCs and HCC1937 CSCs were induced and cultured in DMEM-F12 (Gibco, Thermo Fisher Scientific), containing 2% B27 (Gibco), b-FGF 10 μg/L (Promega), EGF 20 μg/L (Promega), as previously described. All cells were incubated in a 5% CO2 air at 37 °C. The shRNA expressing lentivirus for HES1 and the Slug-overexpression lentivirus were purchased from Hanheng Company (Wuhan, China) and GeneChem Company (Shanghai, China). The sequences of HES1-shRNA used to knockdown HES1 were GGACATTCTGGAAATGACA. Puromycin (10 mg/ml; Sigma, USA) was used to select stably transfected cells. Human Slug cDNA was amplified by PCR and cloned into pcDNA3.1 plasmid. Triple-negative cancer cells were transfected with Slug plasmid by Lipofactamine 3000 (Invitrogen, Carlsbad, CA, USA).
7
Breast Cancer Cell Line Cultivation and Hypoxia
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MCF7 and MDA-MB-231 human breast carcinoma cell lines were obtained from ATCC (Manassas, VA, USA). MCF7 and MDA-MB-231 cells were maintained in high-glucose (4.5 mg/ml) DMEM (HyClone) or L15 (Gibco) medium with 10% (v/v) FBS (HyClone). MCF7 mammosphere (MCF7 MS) cell lines were cultured in DMEM-F12 medium supplemented with 2% B27 (Gibco, Thermo Fisher Scientific), b-FGF 10 μg/L (Promega), EGF 20 μg/L (Promega). For hypoxia treatment, MCF7 cells were cultured under continuous reduced oxygen conditions (1%) using a HERACELL 150i CO2 Incubator (Thermo Fisher Scientific Inc). Dickkopf-1 (DKK-1) was purchased from StemRD. Paclitaxel, L685458 and PEG-35 castor oil were obtained from Sigma.
8
Mammosphere Formation Assay Protocol
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The mammosphere formation assay was performed as described in a previous study with few modifications. Following puromycin selection, the stably infected single cells were suspended in DMEM/F12 (HyClone) supplemented with epidermal growth factor (EGF, 20 ng/mL, Promega), basic fibroblast growth factor (bFGF, 10 ng/mL, Promega), and 2% B27 (Gibco, Thermo Fisher Scientific) and seeded (2000 cells/well) in 6‐well ultra‐low adhesion plates (Corning). The cells were cultured for 7‐14 days and the spheres were imaged. Spheres larger than 100 µm were counted.
9
Breast Cancer Cell Line Culturing
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Breast cancer cell lines MCF-7 and T47D gained from the American Type Culture Collection (ATCC) were cultured by high-glucose (4.5 mg/ml) DMEM (HyClone, USA) with 10% serum (Tianjin Hao Yang Biological Manufacture CL, China). MCF-7 mammosphere (MCF-7 MS) and T47D mammosphere (T47D MS) were inducted and cultured by DMEM/F12 (Gibco) with EGF 20 μg/L (Promega), b-FGF 10 μg/L (Promega) and 2% B27 (Gibco). All of the cells used in our research were cultured in the condition containing 5% CO2 and 95% air at 37 °C.
10
Mammosphere Formation Assay Protocol
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After treated with PTX for 48 h, MCF7 MS cells (2000 cells/ml) were culture in ultra-low adhesion plates (Corning) in DMEM-F12 (GIBCO), containing 2% B27 (Gibco, Thermo Fisher Scientific), b-FGF 10 μg/L (Promega), EGF 20 μg/L (Promega). After culturing for 14 days, mammosphere with diameter > 150 μm were counted. Six replicate wells were included in each analysis and at least three independent experiments were conducted.
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