All the indicated cell lines were purchased from the American Type Culture Collection. PC3 cells were cultured in RPMI 1640 medium and Ham's F12 medium (1:1; Gibco) containing 10% FBS (Gibco) and 1% penicillin–streptomycin (Gibco). DU‐145 were cultured in RPMI 1640 medium containing 10% FBS and 1% penicillin–streptomycin. PCa spheres were cultured in serum‐free DMEM/F12 medium (1:1, Gibco) supplemented with 20 ng/ml of basic human FGF (Sigma), 20 ng/ml of EGF (Sigma), 3 μg/ml of Insulin (Sigma), and 1× B27 (Gibco). 1 × 104 cells were seeded into low adhesion petri dishes (Greiner) and cultured for 6–8 days. About 20% of media was changed every 2 days. All cells were regularly tested for mycoplasma contamination. PC3 cells were treated with 1 μM of BAZ2A‐BRD inhibitors BAZ2‐ICR (SML1276, Sigma) and GSK2801 (SML0768, Sigma), EZH2 inhibitor GSK126 (S7061‐5MG, Lubio), BET inhibitors JQ1 (SML1524, Sigma), or DMSO as control. Fresh medium supplemented with the drugs was added every 2 days. PCa spheres were culture for 6 days. Cells were collected and transferred to a 24‐well plate to image and quantify the number of PCa spheres upon drug treatments.
Basic human fgf
Manufactured by Merck Group
Sourced in France
Basic human FGF is a recombinant protein that functions as a growth factor. It is a key regulator of cell growth, differentiation, and survival. This product is suitable for use in various cell culture and biological research applications.
Automatically generated - may contain errors
Lab products found in correlation
Epidermal growth factor (egf), by Merck Group (2 mentions)
Ham s f12 medium, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Insulin, by Merck Group (1 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (1 mentions)
Gsk2801, by Merck Group (1 mentions)
Sml1524, by Merck Group (1 mentions)
Hepes buffer, by Merck Group (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (1 mentions)
Heparin, by Merck Group (1 mentions)
Progesterone, by Merck Group (1 mentions)
Putrescine, by Merck Group (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Collagenase type 4, by Merck Group (1 mentions)
Dmem f12, by Merck Group (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
4 protocols using basic human fgf
1
Prostate Cancer Spheroid Culture and Drug Treatment
2
Clonogenicity Assay for Cancer Stem Cells
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Example 2
The clonogenicity tests were carried out in a 6 well plate at a density of 500 cells/cm2 in a medium composed of MEM (Gibco) supplemented with 50 units/mL of penicillin, 50 unit/mL of streptomycin (Gibco) and 2.4 g/l of sodium bicarbonate, 1 M of HEPES buffer (Sigma Aldrich, Saint-Quentin-Fallavicr, France), 1× progesterone (Sigma Aldrich), 1× putrescine (Sigma), 0.025 g/mL heparin (Sigma Aldrich), 30% (m/v) glucose (Sigma Aldrich), 1× growth supplement B27 (Invitrogen, Carlsbad, Calif.), 20 ng/mL EGF (Sigma Aldrich), 20 ng/mL basic human FGF (Sigma Aldrich), 1× insulin transferrin sodium selenite supplement (Rock diagnostics, Meylan, France).
The development of colonospheres was observed after incubation a 37° C. in a CO2 atmosphere and quantified with the ImageJ® software.
The results are shown on the graph of
The cancer stem cells isolated according to the method described in the present invention lead to the formation of spheres of larger diameter than those of cells isolated with a kit based on the recognition of the marker CD133.
The method according to the present invention therefore makes it possible to obtain stem cells capable of reforming tumors much more effectively than the method currently available and considered as the standard.
3
Differentiation of EB Cells to Endothelial Cells
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Day 3.5 EB cells were replated at a density of 80,000 cells per gelatin-coated 96-well plate, in StemPro34 serum-free medium supplemented with 100 U ml−1 penicillin, 100 μg ml−1 streptomycin (Gico/BRL), 2 mM glutamine (Gibco/BRL), 10 ng ml−1 human basic FGF, 12.5 ng ml−1 human FGF10, 5 ng ml−1 mouse VEGF and 1 mM ascorbic acid (Sigma, A-4544)27 (link). All growth factors were purchased from R&D Systems. Medium was changed every other day until day 7, when cultures were analysed.
4
Cultivation and Harvesting of Human Embryonic Stem Cells
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hESCs (H1 cell line, WiCell, WI, USA) were cultured on MEFs in hESC medium, which is generally used to culture human embryonic stem cells. The medium was composed of DMEM/F12 (Sigma-Aldrich), 20% KnockOut Serum Replacement (Gibco), 1 mM L-glutamine (PAA), 1% non-essential amino acids (PAA), 0.1 mM 2-mercaptoethanol (Invitrogen), 13 mM HEPES (Gibco) and 8 ng/ml human basic FGF (Sigma-Aldrich). To collect hESCs for the microarray analysis, the hESC cell cultures were first treated with collagenase type IV (Sigma-Aldrich) to remove MEFs, and then the hESC colonies were scraped from culture plates using a cell scraper. The scraped hESCs were centrifuged for 5 minutes at 1000 rpm and washed once with PBS. Washed hESCs were lysed using SuperAmp™ Lysis Buffer and stored at −20 °C until further use.
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