GC cells were inoculated in the ultra-low adhesion 24-well plates (Corning, Corning, NY, USA) at a density of 3000 cells per well and incubated with RPMI 1640 medium containing 2% B27 (Thermo Fisher), 20 ng/mL bFGF (Solarbio, Beijing, China), and 20 ng/mL EGF (MCE) for 7 days. To calculate the sphere formation efficiency, spheroids between 40 and 100 µm in size were counted using an inverted microscope (Olympus, Tokyo, Japan).
Basic fibroblast growth factor (bfgf)
Manufactured by Solarbio
Sourced in China
BFGF is a lab equipment product offered by Solarbio. It is a versatile instrument designed for various applications in life science research. The core function of BFGF is to provide a controlled environment for cell culture and biological experimentation.
Automatically generated - may contain errors
Lab products found in correlation
Epidermal growth factor (egf), by Solarbio (4 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Insulin, by Solarbio (2 mentions)
Ultra low adhesion 24 well plate, by Corning (1 mentions)
Inverted microscope, by Olympus (1 mentions)
Trypsin, by Wuhan Servicebio Technology (1 mentions)
Fetal bovine serum (fbs), by Absin (1 mentions)
Penicillin, by Solarbio (1 mentions)
Streptomycin, by Solarbio (1 mentions)
Ultra lowattachment 24 wells plates, by Corning (1 mentions)
Ix53 dp80, by Olympus (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Low attachment 6 well plate, by Corning (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Neurobasal medium, by Thermo Fisher Scientific (1 mentions)
Epidermal growth factor (egf), by Merck Group (1 mentions)
Ultra low attachment 6 well plate, by Corning (1 mentions)
Heparin, by Solarbio (1 mentions)
7 protocols using basic fibroblast growth factor (bfgf)
1
Sphere Formation Assay for GC Cells
2
Isolation and Culture of Spotted Sea Bass Skeletal Muscle Cells
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Healthy spotted sea bass weighing 28.46–31.35 g were anesthetized with 40 mg mL−1 3-aminobenzoic acid ethyl ester methane-sulfonate (MS-222) and then wiped with 75% ethanol. The dorsal white skeletal muscle (1 cm3) was immediately taken and washed four times in phosphate-buffered saline (PBS) with 1% 100 U mL−1 penicillin and 100 µg mL−1 streptomycin (Solarbio, Beijing, China). Next, the skeletal muscle tissues were cleaned by removing adipose tissues, cut into small pieces (1 mm3), evenly distributed into a 175 cm2 standard cell culture chamber, supplemented with growth medium (GM) (L-15 medium (G-CLONE, Beijing, China) mixed with 20% fetal bovine serum (FBS, ABSIN, Shanghai, China), 10 ng mL−1 bFGF (Solarbio, Beijing, China), 1% 100 U mL−1 penicillin and 100 µg mL−1 streptomycin), and then cultured at 25 °C in a CO2-free incubator (Jinghong, Shanghai, China). Every 3–4 days, the whole medium was replaced with fresh medium until cell emigration and passaging. When the primary cells grew to approximately 70% abundance, 0.25% trypsin (Servicebio, Wuhan, China) was used for subculture, and the isolated cell suspension was precultured in the incubator for 2 h to remove the fibroblasts.
3
Ovarian Cancer Tumorsphere Cultivation
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Single-cell suspensions of ovarian cancer cells (4 × 102 cells/mL) were plated on ultra-low attachment 24 wells plates (Corning, America) and cultured in phenol red-free DMEM/F12 (Gibco, America) containing B27 supplement (Gibico, America, #12587010) and 20 ng/mL epidermal growth factor (EGF, Solarbio, China, #P00033), 20 ng/mL basic fibroblast growth factor (bFGF, Solarbio, China, #P00032), and 5 μg/mL insulin (Solarbio, China, #I8040). Tumorsphere were visualized under a phase-contrast microscope (Olympus, America, IX53 + DP80).
4
Triple-Negative Breast Cancer Spheroid Assay
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Human triple-negative breast cancer cell line MDA-MB-231 and mouse triple-negative breast cancer cell line 4T1 were purchased from ATCC and cultured in DMEM medium with 10% fetal bovine serum and was aired with 5% CO2 at 37 °C. Single MDA-MB-231 cells or 4T1 cells were plated in low attachment 6-well plates (Corning, Oneonta, NY, USA) at a density of 5000 cells/well, and cultured in serum-free DMEM/F12k containing B27 (Thermo Fisher Scientific, Inc., Waltham, MA, USA), epidermal growth factor (EGF, 20 ng/mL; Solarbio Science and Technology Co., Ltd., Beijing, China), and basic fibroblast growth factor (bFGF20 ng/mL, Solarbio Science and Technology Co., Ltd., Beijing, China). The cells were allowed to grow for 7 days; then, the medium was changed, and they were cultured for another 7 days. The quantification of the number of spheres was carried out using microscopy to count all spheres with a diameter bigger than 50 μm. Experiments were repeated three times, with three replicates each.
5
Culturing PC12 Cells and Isolating Rat NSCs
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Rat pheochromocytoma (PC12) cells (CL-0412, Wuhan Procell Co., Ltd) were cultured in RPMI-1640 medium (Gibco) with 5% fetal bovine serum (FBS, Gibco), 10% horse serum (HS, Gibco) and 1% penicillin/streptomycin (PS, Gibco). Neural stem cells (NSCs) were isolated from the hippocampus of E13 Sprague-Dawley rat embryos as described previously [1 (link)]. Briefly, the hippocampus was gently extracted, stripped of blood vessels, cut into small pieces, and digested with 0.05% EDTA/trypsin (Gibco, USA) at 37 °C for 10 min, followed by centrifugation at 200×g for 5 min. Cells were cultured in proliferative NSC medium containing Neurobasal medium (Gibco, USA), 2% B27 (Gibco, USA), 1% PS (Sigma-Aldrich, USA), 1% glutamax (Gibco, USA), 20 ng/mL basic fibroblast growth factor (bFGF, Solarbio, China), and 20 ng/mL epidermal growth factor (EGF, Solarbio, China) in a 5% CO2 humidified 37 °C incubator. For further experiment, 1 × 105 PC12 cells or 1 × 106 were seeded on each sample, respectively. To make sure the cell adhering on the hydrogel surface, the 1 mL cell suspension was dropped onto the hydrogel surface, and after 30 min later the cell culture medium was gently injected into the well from the sidewall to immerse the hydrogel fibers.
6
Tumorsphere Culture Protocol
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For tumorsphere cultures, the isolated cells at 3000 cells/well were seeded in ultralow attachment 6-well plates (Corning, NY) and cultured for 10 days instem cell medium. RPMI-1640 (Gibco) medium containing 20 ng/ml EGF (Millipore), 20 ng/mL bFGF (Solarbio), B27 (1:50,Gibico) and 4 µg/mL insulin (Solarbio). The cells were exposed to fresh medium every 4 days. The number of spheres (defined as diameter ≥ 70μm) was counted under the microscope, and the sphere-forming efficiency was calculated based on the number of initially seeded cells.
7
Mammosphere Formation Assay for Breast Cancer Stem Cells
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Single cells were seeded into the ultralow attachment 24‐well plate (Corning, NY, USA) at a density of 2 × 103 per well with serum‐free DMEM/F‐12 medium (Gibco) supplemented with 1 × B27 (Gibco), 0.4% bovine serum albumin (Solarbio), 0.5 µg mL−1 hydrocortisone (Solarbio), 4 µg mL−1 heparin (Solarbio), 4 µg mL−1 insulin (Solarbio), 20 ng mL−1 EGF (Solarbio), and 20 ng mL−1 bFGF (Solarbio). Cells were cultured for 5–7 days with 500 µL fresh medium added every 3 days, and spheres (> 50 µm) were then counted and photographed using a light field microscope equipped with a phase‐contrast module (Leica, Wetzlar, Germany). Mammosphere formation efficiency (MFE) was calculated as follows: MFE (%) = (the number of mammospheres identified per well/the number of cells seeded per well) × 100%. For the limiting dilution analysis of the mammosphere formation, single cells were seeded into the Corning Costar ultralow attachment 96‐well plate at the density of 1–2 × 102 per well. After 5–7 days, the percentage of wells without spheres was plotted against the number of cells per well, and the regression lines were generated accordingly.
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