FACS-sorted MMTV-Wnt1 tumor cells were cultured in the presence of vehicle or calcitriol in DMEM/F12 medium with 2% B27, 20 ng/ml mouse EGF, 20 ng/mL human FGF (BD, #354060), and 1% antibiotics, and plated on top of solidified Matrigel (28 (link)) for 10–14 days. Spherical colonies (> 50 μm in diameter) were counted using Clono-counter or manually. For the secondary colony formation assay, spheroids were dissociated with 1 mg/mL dispase (Invitrogen, #17105-041) for 30 minutes, digested with trypsin/0.05% EDTA for 5 minutes, and passaged through a 27-G needle five times to dissociate into single cells (29 (link)). After centrifugation, cells were resuspended and cultured as described above. For radiosensitization experiments, sorted TICs were irradiated by 2 Gy and cultured in media containing vehicle or calcitriol for 10–14 days.
Human fgf
Manufactured by BD
Human FGF is a recombinant protein that functions as a fibroblast growth factor. It is a laboratory reagent used for cell culture and research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Dispase, by Thermo Fisher Scientific (2 mentions)
Mouse egf, by BD (2 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Y 27632, by Merck Group (1 mentions)
Human egf, by R&D Systems (1 mentions)
P3932, by Merck Group (1 mentions)
N2 supplement, by Thermo Fisher Scientific (1 mentions)
B27 supplement, by Thermo Fisher Scientific (1 mentions)
3 protocols using human fgf
1
Mammary Tumor Sphere Formation Assay
2
Culturing Tumor Cells on Matrigel
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FACS-sorted tumor cells were suspended in the culture medium consisting of DMEM/F12 (Invitrogen), 20 ng/mL of mouse EGF (BD, #354001), 20 ng/mL of human FGF (BD, #354060), 10 μM of Y-27632 (Sigma, #Y0503) and 1x B27 (Invitrogen, #17504-044), and they were plated on top of solidified matrigel (BD Bioscience, #356237). Then, 100 μL of the cell suspension with 4k cells was put onto the matrigel, and the plate was incubated at 37°C. After 10-14 days, colonieswere counted under an inverted light microscope. To passage, colonies were dissociated with 1 mg/mL of dispase (Invitrogen, #17105-041) and treated with trypsin/0.05 % EDTA (Gibco, #25200) to a produce single cell suspension [27 (link)].
3
Sphere Forming Capacity Assay
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For detecting sphere forming capacity, cells were resuspended as single cell suspension in serum-free DMEM/F12 medium (Gibco, 31331), containing 1% methylcellulose (Sigma, M0512), 20 ng/ml human EGF (R&D Systems, 236-EG), 20 ng/ml human FGF (BD biosciences, 354060), B27 supplement (1:50, Invitrogen, 17504), N2 supplement (1:100, Gibco, 17502), and 1% P/S. 500 single cells were seeded into individual wells of a poly(2hydroxyethylmetacrylate)-coated (Sigma, P3932) 96-well plates. Colonies with a diameter of >80 µm were counted after 12 days.
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