Basic fibroblast growth factor (bfgf)

Manufactured by Merck Group

Sourced in United States, Germany, United Kingdom, China, Canada, Japan, France, Italy, Switzerland, Israel, Spain

BFGF is a laboratory equipment product manufactured by Merck Group. It is a device used for the purification and isolation of recombinant basic fibroblast growth factor (bFGF) from bacterial or mammalian cell cultures. The core function of BFGF is to facilitate the extraction and concentration of this important biomolecule for further research and development applications.

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Basic fibroblast growth factor (bFGF; Fibroblast growth factor (bFGF, Fibroblast growth factor-basic

739 protocols using basic fibroblast growth factor (bfgf)

Neural Induction and Maintenance of NESCs

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IVF3.2 and IVF3.3 rESCs and monkey fibroblast-derived iPS line 1.1 were cultured on X-ray-inactivated CF-1 mouse embryonic φμbroblasts (MEFs) in ESCs growth media (DMEM/F12 [1:1] [Invitrogen] containing 15% KSR [Invitrogen] and 5 ng/mL bFGF [Millipore]) (Chen et al., 2015 (link), Li et al., 2005 (link), Sun et al., 2011 (link)).
ESCs or iPS1.1 were digested with Collagenase IV (Gibco), and neural induction was induced by switching from ESC growth media to differentiation media in suspension culture (Advance DMEM/F12 [1:1] [Invitrogen]: Neurobasal media [Invitrogen] [1:1 mixture] supplemented with 1 × N2 [Invitrogen], 1 × B27 [Invitrogen], 10 ng/ml bFGF [Millipore], 3 μM CHIR99021 [Cellagen Technology], 5 μM SB431542 [Cellagen Technology], 0.2 μM compound E, and 0.1 μM LDN193189 [Cellagen Technology]). After 6 days, EBs were transferred to 5 μg/ml laminin (Gibco)-coated plates for attachment culture, and the media were switched to NESCs culture media (Neurobasal media, including B27, N2, and NEAA [Sigma], 1% Glutmax [Sigma], 3 μM CHIR99021, 5 μM SB431542, 10 ng/ml bFGF, and 1,000 U/ml hLIF [Millipore]). To encourage cell propagation, 0.025% trypsin was used to digest NESCs when passaging. NESCs were routinely passaged to 1:8 to 1:16 ratios every 3 to 4 days. For NT formation, NESCs were continually cultured 8 to 10 days before passaging.

Zhu X., Li B., Ai Z., Xiang Z., Zhang K., Qiu X., Chen Y., Li Y., Rizak J.D., Niu Y., Hu X., Sun Y.E., Ji W, & Li T. (2015). A Robust Single Primate Neuroepithelial Cell Clonal Expansion System for Neural Tube Development and Disease Studies. Stem Cell Reports, 6(2), 228-242.

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Neurosphere Culture and Differentiation

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Neurosphere cultures were prepared as described by Imura et al. [23 (link)]. Briefly, mouse pups of age postnatal day 1 were sacrificed by decapitation and the brains were quickly removed into chilled HEPES solution and dissected by surgical blade. Dissected brains were incubated with digestion buffer and then neutralized with 10% heat-inactivated fetal bovine serum (Invitrogen). Cells were dissociated by gentle mechanical pipetting and resuspended at 2 × 10⁵ cells/mL in serum-free DMEM/F12 medium (Gibco-BRL), which is supplemented with N2 (Gibco-BRL), basic fibroblast growth factor (bFGF, 20 μg/mL; Sigma-Aldrich), and epidermal growth factor (EGF, 10 μg/mL; Sigma-Aldrich). Cells were incubated at 37°C with 5% CO₂ for 6 days to obtain the primary neurospheres and growth factors were added every 3 days. In the AMD-treated group, AMD were dissolved in culture medium and added every 3 days. To evaluate differentiation ability, 6 days in vitro (DIV) neurospheres were plated onto 0.1% fibronectin- (Sigma-Aldrich) coated dishes in bFGF/EGF-free neurobasal medium supplemented with N2 for 4 days.

Ho S.Y., Ling T.Y., Lin H.Y., Liou J.T., Liu F.C., Chen I.C., Lee S.W., Hsu Y., Lai D.M, & Liou H.H. (2017). SDF-1/CXCR4 Signaling Maintains Stemness Signature in Mouse Neural Stem/Progenitor Cells. Stem Cells International, 2017, 2493752.

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Tumorsphere Formation Assay Protocol

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A total of 500 SP and non-SP (NSP) cells were plated onto a 24-well ultra-low attachment plate, and cultured in a DMEM/F12 serum-free medium (Gibco^®; Thermo Fisher Scientific, Inc.) supplemented with 4 µg/ml insulin (Sigma-Aldrich), 10% human leukocyte antigen B27 (Gibco^®; Thermo Fisher Scientific, Inc.), 20 ng/ml epidermal growth factor (EGF; Sigma-Aldrich), and 20 ng/ml basic fibroblast growth factor (bFGF; Sigma-Aldrich), for 10 days. Tumorspheres >50 mm in diameter were counted under a phase-contrast microscope (IX50; Olympus Corporation, Tokyo, Japan).

WEI Z., LV S., WANG Y., SUN M., CHI G., GUO J., SONG P., FU X., ZHANG S, & LI Y. (2016). Biological characteristics of side population cells in a self-established human ovarian cancer cell line. Oncology Letters, 12(1), 41-48.

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Mammosphere Formation and Clonogenic Assay

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For mammosphere formation assay, cells were seeded into ultralow attachment plates (Corning) at a density of 20,000 viable cells/mL in a serum-free DMEM-F12 supplemented with 100× insulin, 20 ng/mL epidermal growth factor and 20 ng/mL basic fibroblast growth factor (Sigma), and 0.4% bovine serum albumin (Sigma), 100 × penicillin &streptomycin for two weeks until the mammosphere became visible.
For clonogenic assay, cells were seeded onto 35 mm² dishes at a density of 5000 viable cells/mL in a serum-free DMEM-F12 supplemented with 100× insulin, 20 ng/mL epidermal growth factor and 20 ng/mL basic fibroblast growth factor (Sigma), and 0.4% bovine serum albumin (Sigma), 100 × penicillin &streptomycin for two weeks until the colonies became visible and stained with Crystal Violet Staining Solution.

Niu Y., Lin Z., Wan A., Chen H., Liang H., Sun L., Wang Y., Li X., Xiong X.F., Wei B., Wu X, & Wan G. (2019). RNA N6-methyladenosine demethylase FTO promotes breast tumor progression through inhibiting BNIP3. Molecular Cancer, 18, 46.

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Photoreceptor Differentiation of phDPSCs

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phDPSCs were induced toward a photoreceptor fate as described previously (15 (link)). Briefly, cells were seeded on laminin-coated plates, allowed to expand for three days before being switched to Neurobasal A media (Gibco, Thermo Fisher Scientific) supplemented with B27 (1:50, Thermo Fisher Scientific), epidermal growth factor (20 ng/mL, Sigma-Aldrich) and basic fibroblast growth factor (40ng/mL, Sigma-Aldrich). On day 8 of differentiation, this was changed to 50% DMEM (Gibco, Thermo Fisher Scientific) + 50% F12 (Gibco, Thermo Fisher Scientific) media supplemented with insulin-transferrin-sodium selenite (conc, Gibco, Thermo Fisher Scientific) and basic fibroblast growth factor (40 ng/mL, Sigma-Aldrich). From day 15 to day 21 of differentiation, retinoic acid (0.5 μg/ml, Sigma-Aldrich) was added to the culture medium. All differentiation media contained penicillin (50 μg/ml, Gibco, Thermo Fisher Scientific) and streptomycin (500 μg/ml, Gibco, Thermo Fisher Scientific) to prevent bacterial contamination. Cells were photographed/lysed to isolate RNA or fixed for immune-cytochemistry at weekly time points.

Balasankar A., Chan S.Y., Babu V.P., Yam G., Tin G.B, & Singhal S. (2022). Dental pulp stem cells retain mesenchymal phenotype despite differentiation toward retinal neuronal fate in vitro. Frontiers in Medicine, 9, 821361.

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Mammosphere Culture Protocol

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Cells were plated in ultra-low-attachment six-well plates (Corning) and cultured in DMEM/F12 serum free medium (Gibco), supplemented with B27 (1:50, Invitrogen), 20 ng/mL basic fibroblast growth factor (bFGF, Sigma), 20 ng/mL human epidermal growth factor (Sigma), 5 μg/m Insulin, 0.4% Bovine Serum Albumin (BSA), at 37 °C in an atmosphere of 5% CO₂. The number and volume of the mammospheres were photographed under an inverted microscope (Nikon).

Zhu Z., Yuan J., Xu X., Wei Y., Yang B, & Zhao H. (2020). Eucannabinolide, a novel sesquiterpene lactone, suppresses the growth, metastasis and BCSCS-like traits of TNBC via inactivation of STAT3. Neoplasia (New York, N.Y.), 23(1), 36-48.

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Primordial Germ Cell Purification and Culture

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The cells were purified as follows:

bPGCs: ACK treatment or Percoll

gPGCs: trypsin or trypsin and Percoll

After purification, the cells were transfected using two methods: electroporation at 200 V and 900 μF for 32 ms or lipofection with Xtreme (Roche) and 20 μg pEGFP-N1. Next, they were seeded into 4-well plates with a density of 5 × 10⁵ cells and over a period of 24 h, the PGCs were grown in OptiMEM I supplemented with antibiotics at 37 °C and 5 % CO₂ in air. After 24 h, the medium was replaced with OptiMEM I C [C: supplemented with 2 % chicken serum (Gibco), 10 % FBS (Sigma), 20 ng/ml bFGF, basic fibroblast growth factor (Sigma), 9 ng/ml mLIF, murine leukaemia inhibitory factor (Sigma), 5 ng/ml hSCF, human stem cell factor (Sigma), antibiotics and G418 (50 μg/ml, Sigma)]. Every 3 days, the medium was changed.

Chojnacka-Puchta L., Sawicka D., Lakota P., Plucienniczak G., Bednarczyk M, & Plucienniczak A. (2015). Obtaining chicken primordial germ cells used for gene transfer: in vitro and in vivo results. Journal of Applied Genetics, 56(4), 493-504.

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Isolation and Culture of Glioma Stem-like Cells

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The HEB, U251MG and U87MG cell lines were purchased from the Chinese Academy of Sciences Cell Bank [18 (link)]. The authenticity of cancer cell lines was tested by short tandem repeat profiling. All cell lines were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS (Gibco, USA). Glioma stem-like cells (GSCs) were cultured and isolated from U251 and U87MG glioma cell lines by using serum-free medium (SFM) which was supplemented with 20 ng/mL basic fibroblast growth factor (bFGF; Sigma-Aldrich, USA), 20 μL/mL B27 supplement (Life Technologies), and 20 ng/mL EGF (Sigma-Aldrich, USA). The medium was refreshed every 2 days. After 10 days, the tumor spheres diameters larger than 50 μm were counted and photographed using phase contrast microscope [19 (link)]. The number of Sphere Forming Units (SFU) is calculated according to the following formula: SFU = (number of spheres counted/number of input cells) × 100.

Wang Z., Xu X., Liu N., Cheng Y., Jin W., Zhang P., Wang X., Yang H., Liu H, & Tu Y. (2017). SOX9-PDK1 axis is essential for glioma stem cell self-renewal and temozolomide resistance. Oncotarget, 9(1), 192-204.

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Isolation and Culture of BMMSCs and ADMSCs

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BM mononuclear cells (BMMNCs) were obtained by conducting equal-volume Ficoll centrifugation (2000 rpm, 40 minutes) (GE Healthcare, UK). The BMMNCs were then seeded in a flask at a cell density of 2×10⁶/cm². The basic growth medium consisted of α-MEM (GIBCO, CA, USA)+20% FBS (Corning, NY, USA)+4 ng/mL of bFGF (Millipore, MA, USA)+1xPS (GIBCO, CA, USA). The paired subcutaneous adipose tissues were dissected into small pieces and digested with collagenase IV (SERVA Electrophoresis GmbH, Heidelberg, Germany) and hyaluronidase (Sigma-Aldrich, MO, USA), and were cultured in the basic growth medium (α-MEM+20% FBS+4 ng/mL bFGF+1xPS) at 37°C for 50 minutes with intermittent shaking. The resulting suspensions were filtered using a 70-µm strainer (BD Bioscience, NJ, USA) to remove debris and then centrifuged at 2000 rpm for 10 minutes. The supernatants were discarded, and the cell pellets were resuspended in the basic growth medium and cultured at density of 1.3×10⁵/cm² in a tissue flask (37°C, 5% CO₂). The basic growth medium was changed every 3 days and the cells were maintained at subconfluent levels (Passage 0, P0). The attached cells (BMMSCs and ADMSCs) were then harvested using trypsin-EDTA (GIBCO, CA, USA), and were subcultured at a density of 2–3×10³ cells/cm² under the same conditions used in the primary culture (Passage 1, P1).

Lin H.H., Hwang S.M., Wu S.J., Hsu L.F., Liao Y.H., Sheen Y.S., Chuang W.H, & Huang S.Y. (2014). The Osteoblastogenesis Potential of Adipose Mesenchymal Stem Cells in Myeloma Patients Who Had Received Intensive Therapy. PLoS ONE, 9(4), e94395.

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Oxygen-Glucose Deprivation of iSCs

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In addition, iSCs were subjected to oxygen–glucose deprivation (OGD) treatment as described previously [13 (link),20 (link)]. In brief, iSCs (5 × 10⁴ cells/well) were plated on 12-well dishes (Iwaki, Tokyo, Japan) in a medium containing DMEM/F12, bFGF, EGF, 10% FBS, and N2. One day after plating, the medium was removed and replaced with glucose-free DMEM, FBS (2%), bFGF, and 0.025 μg/mL recombinant human leukemia inhibitory factor (LIF; Millipore). The iSCs were then incubated under hypoxia (1% O₂) for 3 days using a hypoxia-inducing system (Bionix, SUGIYAMA-GEN, Tokyo, Japan). As a control, iSCs were incubated under normoxia for 3 days in glucose-containing DMEM (DMEM/F12), FBS (2%), bFGF, and 0.025 μg/mL LIF (Millipore).

Tatebayashi K., Tanaka Y., Nakano-Doi A., Sakuma R., Kamachi S., Shirakawa M., Uchida K., Kageyama H., Takagi T., Yoshimura S., Matsuyama T, & Nakagomi T. (2017). Identification of Multipotent Stem Cells in Human Brain Tissue Following Stroke. Stem Cells and Development, 26(11), 787-797.

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