Human CD34+, CD34−, or both CD34+ and CD34− cells isolated from HUCB were cultured in methylcellulose-containing medium, H4236 (StemCell Technologies, Vancouver, Canada), supplemented with 20 ng/mL stem cell-derived factor (Kirin, Tokyo, Japan), 50 ng/mL vascular endothelial growth factor (VEGF; R&D Systems, Minneapolis, MN, USA), 20 ng/mL interleukin (IL)-3 (Kirin), 50 ng/mL basic fibroblast growth factor (bFGF; Wako, Osaka, Japan), 50 ng/mL epidermal growth factor (EGF; Wako), 50 ng/mL insulin-like growth factor (IGF)-1 (Wako), 2 U/mL heparin (Ajinomoto, Tokyo, Japan), and 10% fetal bovine serum (FBS; Life Technologies, Carlsbad, CA) on a 35-mm dish (Thermo SCIENTIFIC, Rockford, IL) for 8 d. The cell density of each sample was 5×102 cells per dish or was adjusted depending on the assay. The EPCs were identified as small EPC-CFUs or large EPC-CFUs by visual inspection using a light microscope (OLYMPUS, Tokyo, Japan) under 40x magnification. Small EPC-CFUs were composed of round adhesive cells, and large EPC-CFUs were composed of spindle-shaped cells. Nonattached cells were isolated as small EPCs by washing with PBS (WELGENE, Daegu, Korea), while attached cells were harvested as large EPCs by treatment with 5 mM EDTA (Sigma-Aldrich, St. Louis, MO) in PBS (5 mmol/L) for 5 min at 37°C.
Basic fibroblast growth factor (bfgf)
Manufactured by Fujifilm
Sourced in Japan, United States, Canada
The BFGF is a specialized laboratory equipment designed for conducting various biological and biochemical analyses. It serves as a versatile tool for researchers and scientists in the field of life sciences. The core function of the BFGF is to facilitate the efficient separation and purification of biomolecules, such as proteins, nucleic acids, and other macromolecules, through a process known as gel filtration chromatography.
Automatically generated - may contain errors
Lab products found in correlation
Knockout serum replacement (ksr), by Thermo Fisher Scientific (13 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (11 mentions)
Epidermal growth factor (egf), by Fujifilm (9 mentions)
Primate es cell medium, by ReproCELL (8 mentions)
Non essential amino acids (neaa), by Merck Group (8 mentions)
B27 supplement, by Thermo Fisher Scientific (8 mentions)
L glutamine, by Thermo Fisher Scientific (8 mentions)
Vascular endothelial growth factor (vegf), by R&D Systems (6 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (6 mentions)
Dmem f12, by Merck Group (6 mentions)
Y 27632, by Fujifilm (6 mentions)
Collagenase 4, by Thermo Fisher Scientific (6 mentions)
L glutamine, by Merck Group (5 mentions)
Sb431542, by Merck Group (5 mentions)
Penicillin, by Thermo Fisher Scientific (5 mentions)
Streptomycin, by Thermo Fisher Scientific (5 mentions)
Activin a, by R&D Systems (4 mentions)
Human transferrin, by Roche (4 mentions)
Mtesr1, by STEMCELL (4 mentions)
Primate es medium, by ReproCELL (4 mentions)
91 protocols using basic fibroblast growth factor (bfgf)
1
Isolation and Characterization of EPCs from HUCB
2
Cytokine and Growth Factor Effects on Colonic Epithelial Cells
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LS-174T and HT-29 human colonic epithelial cells were grown in RPMI 1640 medium (Nakarai Tesque, Inc., Kyoto, Japan) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin (Wako) [23] . Human interleukin (IL)− 6 and tumor necrosis factor (TNF)α were purchased from Roche Diagnostics (Indianapolis, IN), and human IL-8, IL-17A, IL-22, hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF) were from Wako. Cells were treated with 20 ng/mL IL-6, 10 nM IL-8, 1 µg/mL IL-17A, 20 ng/mL IL-22, 20 ng/mL TNFα, 50 ng/mL HGF, 10 nM bFGF, and 10 nM EGF for 24 h.
3
Endothelial Progenitor Cell Colony Assay
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Selected cell lines were cultured in methylcellulose-containing medium (M3434, StemCell Technologies, Vancouver, BC, Canada) with 50 ng/mL vascular endothelial (VE) growth factor (R&D Systems, Minneapolis, MN, USA), 50 ng/mL basic fibroblast growth factor (Wako, Osaka, Japan) and 10% fetal bovine serum (FBS) on 35-mm dishes. Cell densities were 1×103 for each cell lines. Dishes were plated in triplicate and kept in humidified incubator for 8–10 days until colonies started to develop. EPC colony forming units (CFUs) were defined as cluster-like collections of cells associated with attached spindle-shaped cells. The EPC-CFUs were identified by visual inspection with an inverted microscope under 20-40× magnification. Images were acquired on a Zeiss Observer. A1 (Zeiss Microscopy, Thornwood, NY) microscope using AxioVision 4.6.3 software (Zeiss Microscopy, Thornwood, NY).
4
Establishment of Retinal Pigment Epithelium
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The hiPS cell lines 454E2 [12 (link)] and 253G1 [13 (link)], which were derived from healthy human dental pulp cells using six transcription factors (Oct3/4, Sox2, Klf4, L-Myc, Lin28, and p53) and dermal fibroblast cells using three transcription factors (Oct3/4, Sox2, and Klf4), respectively, were supplied by RIKEN BioResource Center (Ibaraki, Japan). HfRPE and ARPE19 were purchased from Lonza (Basel, Switzerland) and the ATCC (Manassas, VA, USA), respectively. The methods used for hiPSC maintenance and differentiation have been previously described [14 (link)]. RPE were cultured on CELLstart-coated (GIbcO, Carlsbad, CA, USA) dishes in preconfluent medium (F10 (Sigma-Aldrich Corp., St. Louis, MO, USA) with 10% fetal bovine serum) before reaching confluence and in postconfluent medium (DMEM/F12 (7 : 3) supplemented with B27 (Invitrogen, Carlsbad, CA, USA), 2 mM L-glutamine (Sigma-Aldrich Corp.), 10 ng/mL basic fibroblast growth factor (Wako, Osaka, Japan), and SB431542 (0.5 μM, Sigma-Aldrich Corp.)) after reaching confluence. The medium was changed every 2 to 3 days. Cultured RPE were recorded using a laser scanning confocal microscope (IX81; Olympus, Tokyo, Japan).
5
Mammary Epithelial Sphere Formation
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A total of 5 × 103 NMuMG‐mock, NMuMG‐GPNMB(WT), or NMuMG‐GPNMB(ΔKLD) cells were cultured in DMEM/F12 medium (Sigma‐Aldrich) supplemented with 20 μL/mL B27 (Invitrogen), 20 ng/mL EGF (Sigma‐Aldrich), and 20 ng/mL basic fibroblast growth factor (Wako Pure Chemical Industries) in each ultra‐low attachment culture dish (35 mm; Corning). The size of the spheres was measured and the number of the spheres was counted on day 7.
6
Culturing Human Brain Tumor Cell Lines
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Human brain tumor cell lines (U87, U138, U251, and T98G) were purchased from the American Type Culture Collection (Manassas, VA, USA). These cell lines were cultured in DMEM (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum and antibiotics under a humidified atmosphere containing 5% CO2 at 37 °C. Patient glioblastoma cells were first cultured in DMEM/F-12 medium (Thermo Fisher Scientific) containing B27 supplement minus vitamin A (Thermo Fisher Scientific), epidermal growth factor, and basic fibroblast growth factor (20 ng/ml each; Wako Pure Chemical Industries, Osaka, Japan) to generate glioblastoma stem-like cells as neurospheres. These glioblastoma stem-like cells were then cultured in DMEM supplemented with 10% fetal bovine serum (FBS) to generate patient-derived glioblastoma cell lines or with all-trans retinoic acid (RA) for differentiation. To generate CD1d-transfected U87 cells, pCMV6-XLA4/hCD1d (OriGene Technologies, Rockville, MD, USA) was transfected into U87 cells using a cationic lipid-based transfection reagents, Lipofectamine 3000 (Thermo Fisher Scientific), according to the respective manufacturer’s instructions. Mock group cells were mock-transfected with Lipofectamine 3000 reagent only.
7
Culturing Bladder Cancer and Endothelial Cells
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The human bladder carcinoma cell line T-24 (EJ-1 strain) and its transfectants (Mock-T24 and γ2SA-T24) were used in our previous study.(28 (link)) These cells were maintained in DMEM/F12 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FCS (Nichirei Biosciences, Tokyo, Japan) and antibiotics. Human umbilical vein endothelial cells were purchased from Kurabo (Osaka Japan) and maintained on type I collagen (40 μg/mL) (Nitta Gelatin, Osaka, Japan) coated plates in MCDB131 medium (Sigma-Aldrich) supplemented with 10 ng/mL EGF (Wako, Osaka Japan), 5 ng/mL basic fibroblast growth factor (Wako), 50 μg/mL heparin (Wako), 10% FCS, and antibiotics.
8
Tumor Sphere Formation Assay
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Cells were plated as single cell suspensions in 24-well ultra-low attachment plates at 500 cells/mL density to obtain single cell-derived tumor spheres after siRNA treatment for 48 h. Cells were grown in DMEM/F-12 medium, 20 ng/mL epidermal growth factor (Sigma-Aldrich Corp.), 20 ng/mL basic fibroblast growth factor (FUJIFILM Wako Pure Chemical Corporation), B27 supplement (Thermo Fisher Scientific Inc.) and 5 mg/L gentamicin (Sigma-Aldrich Corp.). Spheres with a diameter >100 µm were counted after 3 days for the Mi cell line and 5 days for the CMM12 cell line.
9
Culturing Human Colorectal Cancer and iPSCs
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The human colorectal cancer cell line HCT 116 was obtained from the American Type Culture
Collection (Manassas, VA, USA). HCT 116 cells were maintained in Dulbecco’s Modified Eagle
Medium (DMEM; Invitrogen Corporation, Carlsbad, CA, USA) supplemented with 10% fetal
bovine serum (Invitrogen), 2 mM L-glutamine (L-Gln; Nacalai Tesque, Inc., Kyoto, Japan),
50 units/ml of penicillin, and 50 µg/ml of streptomycin (P–S; Nacalai
Tesque, Inc.).
Human iPS cell line 201B7 was provided by the RIKEN BRC (Ibaraki, Japan) through the
National BioResource Project of the Ministry of Education, Culture, Sports, Science and
Technology (MEXT), Japan [19 (link)]. Human iPS cells were
maintained with mitomycin C-treated mouse embryonic fibroblast feeder cells in
DMEM:Nutrient Mixture F-12 (Invitrogen) supplemented with 20% knockout serum replacement
(KSR; Invitrogen), L-Gln, P–S, 100 µM nonessential amino acids
(Invitrogen), 100 µM β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO,
USA), and 5 ng/ml of basic fibroblast growth factor (Wako Pure Chemical Industries, Ltd.,
Osaka, Japan).
Collection (Manassas, VA, USA). HCT 116 cells were maintained in Dulbecco’s Modified Eagle
Medium (DMEM; Invitrogen Corporation, Carlsbad, CA, USA) supplemented with 10% fetal
bovine serum (Invitrogen), 2 mM L-glutamine (L-Gln; Nacalai Tesque, Inc., Kyoto, Japan),
50 units/ml of penicillin, and 50 µg/ml of streptomycin (P–S; Nacalai
Tesque, Inc.).
Human iPS cell line 201B7 was provided by the RIKEN BRC (Ibaraki, Japan) through the
National BioResource Project of the Ministry of Education, Culture, Sports, Science and
Technology (MEXT), Japan [19 (link)]. Human iPS cells were
maintained with mitomycin C-treated mouse embryonic fibroblast feeder cells in
DMEM:Nutrient Mixture F-12 (Invitrogen) supplemented with 20% knockout serum replacement
(KSR; Invitrogen), L-Gln, P–S, 100 µM nonessential amino acids
(Invitrogen), 100 µM β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO,
USA), and 5 ng/ml of basic fibroblast growth factor (Wako Pure Chemical Industries, Ltd.,
Osaka, Japan).
10
Dextran-Induced EPC Colony Formation
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5 × 103 EPCs under a dextran‐free and exposed to 10% dextran for 24 h were applied in methylcellulose‐containing M3236 medium (StemCell Technologies, Vancouver, Canada) with 20 ng/mL stem cell factor (Kirin), 50 ng/mL VEGF (R&D Systems), 20 ng/mL interleukin‐3 (Kirin), 50 ng/mL basic fibroblast growth factor (Wako), 50 ng/mL epidermal growth factor (Wako), 50 ng/mL insulin‐like growth factor‐1 (Wako), and 2 U/mL heparin (Ajinomoto) in a 3 cm‐dish. After 15 days in culture, the number of small or large type EPC colonies in a dish was counted under a phase contrast microscope.
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