In vitro expansion of hCEC was carried out in two steps, using a dual media approach with a maintenance cell culture medium (S medium; M5) in combination with a growth cell culture medium (P medium; M4) [22 (link)]. The latter consisted of Ham’s F12/M199 medium (Gibco, USA), supplemented with 5% FBS, 1 mg/mL insulin, 0.55 mg/mL transferrin, and 0.5 µg/mL sodium selenite (ITS) (Sigma Aldrich, Germany), 0.2 mm ascorbic-2-phosphate (Dako; Agilent Technologies, Santa Clara, CA, USA), 10 ng/mL human basic fibroblast growth factor (Sigma Aldrich, Germany), 10 μm ROCK inhibitor Y-27632, and 1% antibiotic/antimycotic solution. Cells were first left to adhere overnight to the surface coated with FNC Coating Mix (AthenaES) in M5 medium, which was replaced by M4 medium to induce cell proliferation. Upon culture confluency (∼2 weeks), M4 medium was replaced by fresh M5 medium (∼1 week). Cell culture mediums were changed every 2 days. Passages were carried out with a plating cell density of 1 × 104 cells/cm2, as previously recommended [27 (link)], after dissociation of TrypLE Select (Gibco, USA).
Human basic fibroblast growth factor
Manufactured by Merck Group
Sourced in United States, United Kingdom, Macao
Human basic fibroblast growth factor is a recombinant protein that stimulates the proliferation of various cell types, including fibroblasts, endothelial cells, and smooth muscle cells. It is a member of the fibroblast growth factor family and plays a role in wound healing, angiogenesis, and tissue repair.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (11 mentions)
Human epidermal growth factor (hegf), by Merck Group (10 mentions)
Chemically defined lipid concentrate, by Thermo Fisher Scientific (10 mentions)
Hydrocortisone, by Merck Group (8 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Ascorbic acid, by Merck Group (6 mentions)
Ebm 2, by Lonza (6 mentions)
Hepes, by Thermo Fisher Scientific (5 mentions)
Bovine serum albumin (bsa), by Merck Group (5 mentions)
Hepes, by GE Healthcare (5 mentions)
Dexamethasone (dex), by Merck Group (4 mentions)
Fbs gold, by GE Healthcare (4 mentions)
Nucleoside, by Merck Group (4 mentions)
Chicken serum, by Merck Group (4 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
Penicillin, by Thermo Fisher Scientific (4 mentions)
Insulin, by Merck Group (3 mentions)
Epidermal growth factor (egf), by Merck Group (3 mentions)
Fetal bovine serum (fbs), by GE Healthcare (3 mentions)
Cultrex rat collagen 1, by R&D Systems (3 mentions)
44 protocols using human basic fibroblast growth factor
1
In vitro Expansion of Human Corneal Endothelial Cells
2
Dystrophin Gene Deletion in DMD Cells
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HEK293T cells were obtained from the American Tissue Collection Center (ATCC) through the Duke Cell Culture Facility and were maintained in DMEM supplemented with 10% fetal bovine calf serum and 1% penicillin/streptomycin. Immortalized myoblasts70 (link) from a DMD patient harboring a deletion of exons 48–50 (Δ48–50) in the dystrophin gene were maintained in skeletal muscle media (PromoCell) supplemented with 20% fetal bovine calf serum (Sigma), 50 μg/ml fetuin, 10 ng/ml human epidermal growth factor (Sigma), 1 ng/ml human basic fibroblast growth factor (Sigma), 10 μg/ml human insulin (Sigma), 1% GlutaMAX (Invitrogen), and 1% penicillin/streptomycin (Invitrogen). All cell lines were maintained at 37°C and 5% CO2. HEK293T cells were transfected with Lipofectamine 2000 (Invitrogen) with 400 ng of each expression vector according to the manufacturer’s protocol in 24 well plates. Immortalized myoblasts were transfected with 5 micrograms of each expression vector by electroporation using the Gene Pulser XCell (BioRad) with PBS as an electroporation buffer using optimized conditions 28 (link). Transfection efficiencies were measured by delivering an eGFP expression plasmid (pmaxGFP, Clontech) and using flow cytometry. These efficiencies were routinely ≥ 95% for HEK293T and ≥ 70% for the immortalized myoblasts.
3
Dystrophin Gene Deletion in DMD Cells
4
Culturing Brain Microvascular Endothelial Cells
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The human brain microvascular endothelial cell line hCMEC/D3 cells were purchased from the National Infrastructure of Cell Line Resource (Beijing, China). The cells were derived from human brain microvascular endothelium and shared characteristics with the BBB, including the expression of TJ proteins (Weksler et al., 2013 (link)). The cells were cultured in DMEM/high glucose (Hyclone, United States) supplemented with 10% FBS (Gibco, United States), 1% penicillin/streptomycin (Gibco, United States), and 1 ng/ml human basic fibroblast growth factor (Sigma-Aldrich, United States) in 5% CO2 at 37°C. According to the experimental requirements, the cells were plated in 6-well plates, 24-well plates, or 96-well plates, respectively, and treated with different drugs for different durations when the cells reached appropriate confluence. A non-serum medium was used for drug treatments.
5
Pancreatic Tumor Cell Culture Protocol
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Tumor cells were cultured from a primary pancreatic tumor isolated from a 17 week old RT2 AB6F1 male mouse; this mouse showed liver metastasis at the time of euthanasia. To deter growth of normal fibroblasts, the tumor cells were grown in a stem cell medium for several weeks, consisting of Dulbecco's modified Eagle's medium/F12 medium (Life Technologies) supplemented with N-2 supplement (Life Technologies), 20 ng/ml human epidermal growth factor (Life Technologies), 10 ng/ml human basic fibroblast growth factor (Sigma-Aldrich), 4 μg/ml heparin (Sigma-Aldrich), 4 mg/ml bovine serum albumin (Life Technologies), 20 μg/ml human insulin, zinc solution (Life Technologies), and 2.9 mg/ml glucose (Sigma-Aldrich) [46 (link)]. After several passages, the cells were switched to RPMI media with 10% fetal bovine serum. SV40 T-antigen antibody (Abcam), insulin antibody (Cell signaling) and CD88 antibody (Biolegend) were used for flow cytometry analysis. Invasiveness was tested using a matrigel invasion chamber (Corning) with or without addition of recombinant mouse C5a protein (R&D Systems). The invasion assay was repeated three times.
6
Osteogenic Differentiation of hMSCs
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Samples were sterilized in 70% ethanol for 1 h, followed by four washes with phosphate buffered saline (PBS; Sigma, Steinheim, Germany) and overnight incubation in an expansion medium (minimum essential medium alpha, α-MEM; Gibco, Thermo Fisher Scientific, Paisley, UK) supplemented with 10% FBS (fetal bovine serum; South American Origin, Biowest, Nuaillé, France), 1% PSN (5 units/ml of penicillin, 5 mg/ml of streptomycin, 10 mg/ml neomycin; Sigma, St. Louis, MO, USA) and 1 ng/mL human basic fibroblast growth factor (Sigma, Jerusalem, Israel). Normal human bone marrow derived mesenchymal stem cells (hMSCs) were purchased from Lonza (Walkersville, MD, USA). The cells were isolated from bone marrow of a 40-year-old female. The cells used in the experiments were from passage 4. MSCs were seeded at a density of 2.5 × 105 per scaffold and incubated in expansion for 7 days and in osteogenic medium (α-MEM supplemented with 10% FBS, 1% PSN, 50 µM ascorbic acid phosphate (Sigma, Osaka, Japan), 2 mM β-glycerophosphate (Sigma, St. Louis, MO, USA), 10 nM 1,25-dihydroxy-vitamin D3 (Sigma, Jerusalem, Israel) and 10 nM dexamethasone (Sigma, Shanghai, China) for an additional 7 days.
7
Human Brain Endothelial Cell Culture
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Human brain endothelial cells (hCMEC/D3;
kindly provided by Dr. P. O. Couraud, Institute Cochin, Paris, France),
passage 25–35, were used as a representative model for human
BBB. hCMEC/D3 cells were cultured in EBM-2 medium (Lonza, MD) supplemented
with 1 ng/mL human basic fibroblast growth factor (Sigma-Aldrich),
10 mM HEPES, 1% chemically defined lipid concentrate (Gibco, NY),
5 μg/mL ascorbic acid, 1.4 μM hydrocortisone, 1% penicillin–streptomycin,
and 5% of heat-inactivated FBS gold (GE Healthcare Life Sciences,
PA). Human neuroblastoma cells (SH-SY5Y) stably expressing wild-type
human APP695 (kindly provided by Dr. Elizabeth A. Eckman, Biomedical
Research Institute of New Jersey, NJ) were maintained in DMEM supplemented
with 10% FBS, glutamine, penicillin (100 units/mL) and streptomycin
(100 μg/mL), and the selective antibiotic Geneticin at 400 μg/mL
(Gibco).61 (link) Cultures were maintained in
a humidified atmosphere (5%CO2/ 95% air) at 37 °C
and media was changed every other day.
kindly provided by Dr. P. O. Couraud, Institute Cochin, Paris, France),
passage 25–35, were used as a representative model for human
BBB. hCMEC/D3 cells were cultured in EBM-2 medium (Lonza, MD) supplemented
with 1 ng/mL human basic fibroblast growth factor (Sigma-Aldrich),
10 mM HEPES, 1% chemically defined lipid concentrate (Gibco, NY),
5 μg/mL ascorbic acid, 1.4 μM hydrocortisone, 1% penicillin–streptomycin,
and 5% of heat-inactivated FBS gold (GE Healthcare Life Sciences,
PA). Human neuroblastoma cells (SH-SY5Y) stably expressing wild-type
human APP695 (kindly provided by Dr. Elizabeth A. Eckman, Biomedical
Research Institute of New Jersey, NJ) were maintained in DMEM supplemented
with 10% FBS, glutamine, penicillin (100 units/mL) and streptomycin
(100 μg/mL), and the selective antibiotic Geneticin at 400 μg/mL
(Gibco).61 (link) Cultures were maintained in
a humidified atmosphere (5%CO2/ 95% air) at 37 °C
and media was changed every other day.
8
Sarcosphere Formation Assay in Vitro
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Sarcosphere formation assay was performed, according the protocol described by Gibbs et al (7 (link)). Cells were plated at a density of 60,000 cells/well in ultra-low attachment six-well plates (Corning, Inc.) containing serum-free DMEM/F12 medium supplemented with N2, 10 ng/ml epidermal growth factor and 10 ng/ml human basic fibroblast growth factor (both purchased from Sigma-Aldrich). The culture was analyzed for sphere formation daily for 7 days, and cell proliferation was measured by checking the absorbance at 450 nm using a plate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Following 7 days of culturing, the total number of sarcospheres generated by FACS-sorted SP and non-SP cells was quantified by inverted phase contrast microscopy (Eclipse TS100; Nikon Corporation, Tokyo, Japan).
9
Neural Differentiation by Growth Factors
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To induce neural differentiation, the cells were cultured in DMEM/F12 medium supplemented with 2% FBS, 80 ng/ml human basic fibroblast growth factor (Sigma-Aldrich), 30 μM forskolin (Sigma-Aldrich), 1% non-essential amino acids (Gibco), 0.1 mM 2-mercaptoethanol (Gibco) and 1% insulin-transferrin-selenium (Gibco). After 2 weeks of culture, the cell cultures were analysed for the expression of glial marker S100 using immunocytochemistry according to the procedure described above.
10
In Vitro Brain Endothelial Cell Culture
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