MSCs were added into 24-well plates covered by glass, PLLA, and stereocomplexed PLA/TTCP45 and cultured with L-DMEM containing 10% FBS. The density of MSCs was 1 × 104 cells/well. After 24 h, the culture medium was replaced by L-DMEM containing 2% FBS and 10 ng/mL bFGF (MedChemExpress, USA). After 24 h, the medium was replaced by L-DMEM containing 2% FBS, 20 ng/mL bFGF and 20 ng/mL EGF (MedChemExpress, USA) every 3 days for 7 days. Then, cells were fixed with 2.5% glutaraldehyde and incubated in 0.1% Triton X-100 for 15 minutes. After that, cells were incubated with the primary antibody of Tuj1 (Cell Signaling Technology, USA.) and Neuron (Cell Signaling Technology, USA) overnight at 4°C and incubated with secondary antibody (Abcam, UK) at 37°C for 1.5 h. Then, Apotome.2 (ZEISS, Germany) was used to take immunofluorescence images.
Basic fibroblast growth factor (bfgf)
Manufactured by MedChemExpress
Sourced in United States
BFGF is a laboratory reagent used in cell culture research. It functions as a growth factor that stimulates the proliferation and differentiation of various cell types, including fibroblasts and endothelial cells.
Automatically generated - may contain errors
Lab products found in correlation
Epidermal growth factor (egf), by MedChemExpress (7 mentions)
Dmem f12, by Thermo Fisher Scientific (3 mentions)
24 well ultra low attachment plate, by Corning (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (2 mentions)
B27 supplement, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Tgf β1, by MedChemExpress (1 mentions)
Ly2109761, by MedChemExpress (1 mentions)
Insulin, by MedChemExpress (1 mentions)
Leibovitz s l 15 medium, by Merck Group (1 mentions)
Ultra low attachment 6 well plate, by Corning (1 mentions)
Penicillin streptomycin solution, by Solarbio (1 mentions)
1xb27 supplement, by Thermo Fisher Scientific (1 mentions)
Eclipse ti series, by Nikon (1 mentions)
Dulbecco s modified eagle s medium f12 medium, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Apotome 2, by Zeiss (1 mentions)
Mem non essential amino acids solution, by Thermo Fisher Scientific (1 mentions)
B 27 serum free supplement, by Thermo Fisher Scientific (1 mentions)
11 protocols using basic fibroblast growth factor (bfgf)
1
Neuronal Differentiation of Mesenchymal Stem Cells
2
Isolation and Activation of Cancer-Associated Fibroblasts
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Primary CAFs were separated from CCA samples as previously reported [35 (link)]. In brief, CCA tissue samples were cut into pieces and maintained in DMEM (Thermo Fisher Scientific) containing 20% FBS and basic fibroblast growth factor (1 ng/mL, MedChemExpress, USA) at 37 °C with 5% CO2. After removing the unattached cells, myofibroblast-like CAFs at passages 4–8 were adopted. The successful isolation of CAFs was identified by high expression of α-SMA, vimentin, and FAP. Primary CAFs were stimulated with TGF-β1 (20 ng/mL, MedChemExpress) for 48 h. For the inactivation of Smad2/3 pathway, CAFs were treated with 50 μM LY2109761 (MedChemExpress).
3
Isolation and Characterization of Melanoma Stem Cells
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Melanoma stem cells and non-stem cells were sorted from MDA-MB-435 cells in our laboratory, which were performed as previously described14 (link). Briefly, the top 1% ALDH1-positive cells were sorted from MDA-MB-435 cells with flow cytometry. As reported, one marker is not enough to fully characterize melanoma stem cells15 (link). To confirm the sorted melanoma stem cells, the tumorsphere formation assay and tumorigenesis in nude mice of ALDH1-positive cells were conducted. All the data demonstrated that the sorted ALDH1-positive cells were melanoma stem cells14 (link). Melanoma non-stem cells were cultured in Leibovitz’s L-15 medium (Sigma, USA) supplemented with 10% fetal bovine serum at 37 °C with 100% humidified atmosphere. Melanoma stem cells were grown in DMEM/F-12 medium (Invitrogen, USA) supplemented with 20 ng/mL epidermal growth factor (MedChemExpress, USA), 10 ng/mL basic fibroblast growth factor (MedChemExpress), 5 μg/mL of insulin (MedChemExpress), and 2% of B-27 (Invitrogen) at 37 °C in a humidified atmosphere with 5% CO2.
4
Sphere Formation Assay for Cancer Stem Cells
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Sphere formation assays in A549/CDDP and H460/CDDP cells were performed 48 h after transfection. Briefly, cells at a density of 1×103 cells/well were seeded into 6-well ultra-low attachment plates (Corning, Inc.) and were then cultured in spheroid medium, composed of DMEM/Nutrient Mixture F-12 (DMEM-F12; Gibco; Thermo Fisher Scientific, Inc.) containing B-27™ Supplement (Thermo Fisher Scientific, Inc.), 20 ng/ml epidermal growth factor (EGF, MedChemExpress), 20 ng/ml basic fibroblast growth factor (MedChemExpress), and 1% penicillin/streptomycin solution (Beijing Solarbio Science & Technology Co., Ltd.). Following 10 days of culture, the number of formed spheres (diameter, >50 µm) was counted, and images were captured under a light microscope with a magnification of ×200.
5
Tumor Sphere Formation Assay
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A549 and H1650 cells (treatment with or without DPEP2 overexpression) were digested with 0.25% Trypsin-EDTA (Invitrogen; Thermo Fisher Scientific, Inc.), centrifuged with a low centrifugation speed (141 × g for 3 min at 25°C; model no. JW-1016; Anhui Jiawen Instrument Equipment Co., Ltd.), and washed thrice with PBS. The cells were resuspended in Dulbecco's modified Eagle's medium/F12 medium (Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 20 ng/ml Epidermal Growth Factor (MedChemExpress), 20 ng/ml Basic Fibroblast Growth Factor (MedChemExpress), and 1X B-27 supplement (Invitrogen; Thermo Fisher Scientific, Inc.). Cells were incubated at a density of 5×103 cells/well in a six-well ultra-low adhesion plate for 7–10 days at 37°C with 5% CO2. Representative tumor spheres were captured at a magnification of ×20 and quantified under a fluorescence microscope (Eclipse Ti Series; Nikon Corporation).
6
Three-Dimensional Cell Culture Protocol
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The cell pellet was resuspended in three-dimensional cell culture medium, which consisted of the following components: DMEM/F12 (Thermo Fisher Scientific, United States), supplemented with 1% penicillin-streptomycin (Gibco, United States), 10% fetal bovine serum (FBS; Wisent, Canada), 0.5% GlutaMax (Gibco, United States), 0.5% N2 supplement (Thermo Fisher Scientific, United States), 0.5% MEM Non-Essential Amino Acids Solution (Thermo Fisher Scientific, United States), 0.5% B-27® Serum-Free Supplement (Thermo Fisher Scientific, United States), 8 mg/mL EGF (PeproTech, United States), 10 ng/mL bFGF (MCE, MedChemExpress, United States), 10 ng/mL IGF-1 (MCE, MedChemExpress, United States), and 5 ng/mL TGF-beta 3 (MCE, MedChemExpress, United States). The prepared cell suspension was then added to an ultra-low attachment U-bottom 96-well plate (Corning, United States). The cell count in each well was adjusted to be between 300 and 5000 cells. The plate was then placed in a 37°C incubator with a 5% carbon dioxide atmosphere. Each well received 200 μL of the medium, which was changed every 24–36 h.
7
Sphere Formation Assay for Progenitor Cells
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A total of 1000 cells/per well were seeded in an ultralow attachment 96-well plate (Corning, USA) and cultivated with serum-free DMEM/F12 medium (Gibco, USA) containing 20 ng/ml EGF (MedChemExpress, USA), 10 ng/ml b-FGF (MedChemExpress, USA), and 2% B27 (MedChemExpress, USA). After incubation for 10 days, the images of spheres were captured under a microscope.
8
ESCC Spheroid Formation Protocol
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ESCC cells were cultured in ultra‐low attachment 24‐well plates (Corning, Union City, CA, USA) at 1000 cells/well with Dulbecco's modified Eagle's medium/F12 medium supplemented with 1 × B27 (Sigma‐Aldrich, St Louis, MO, USA), 20 ng·mL−1 bFGF (MedChemExpress, Monmouth Junction, NJ, USA), 20 ng·mL−1 epidermal growth factor (MedChemExpress) and antibiotics at 37 °C under a 5% humidified CO2 atmosphere. After 10 days, the number and size of spheroids were evaluated and quantified under a microscope.
9
Gastric Cancer Cell Spheroid Cultivation
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Gastric cancer cells were cultured in ultra‐low attachment 24‐well plates (Corning, Union City, CA, USA) at 1000 cells/well with DMEM/F12 medium supplemented with 1 × B27 (Sigma‐Aldrich, St Louis, MO, USA), 20 ng·mL−1 bFGF (MedChemExpress, Monmouth Junction, NJ, USA), 20 ng·mL−1 EGF (MedChem Express) and antibiotics at 37 °C under a 5% humidified CO2 atmosphere. After 10 days, the number and size of spheroid were evaluated and quantified under a microscope.
10
Expansion and Characterization of Expanded ACY-Treated MSCs
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In an ACY (-) group, MSCs were cultured as previously described (Henrionnet et al., 2017 (link); Samal et al., 2021 (link)). In brief, the culture medium contained low glucose DMEM (Gibco) supplemented with FBS (10%, Hyclone), fibroblast growth factor-basic (bFGF, 1 ng/ml, PeproTech), and penicillin/streptomycin (1%, Gibco). In ACY (+) group, the basal medium for culturing MSCs consisted of low glucose DMEM, FBS, bFGF, penicillin/streptomycin, and ACY cocktails consisted of A-83–01 (MedChemExpress), CHIR99021 (Selleck), and Y-27632 (Selleck). The medium was changed 1 day after seeding and every 2 days thereafter. There were 2 μM A-83–01, 3 μM CHIR99021, and 2 μM Y-27632 chosen as the optimal concentration in the ACY (+) group. When cells were approximately 90% confluent, they would be passaged with a split ratio of 1:3.
MSCs at passage 2 (P2) were seeded, without (ACY-) or treated (ACY+) with A-83–01, CHIR99021, and Y-27632, and were continuously cultured to passage 10 (P10;Supplementary Figure S1 ). From passage 5 (P5) to P10, the MSCs were used in subsequent analysis.
MSCs at passage 2 (P2) were seeded, without (ACY-) or treated (ACY+) with A-83–01, CHIR99021, and Y-27632, and were continuously cultured to passage 10 (P10;
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