Three independent glioma initiating cells (GICs) termed TGS-01, TGS-04 and TGS-05 were established as described previously17 (link). All human materials and protocols used in this study were approved by the ethics committee of the University of Tokyo Hospital (24-69-250809) and Medical Mycology Research Center (MMRC), Chiba University (#10). All methods were performed in accordance with each university’s guideline and regulation. Informed consent was obtained from all patients. GICs were passaged in DMEM/F12 serum-free medium (Thermo Fisher Scientific) supplemented with B27 (Thermo Fisher Scientific), 20 ng/ml of EGF, and 20 ng/ml of bFGF (both from PeproTech) using ultra-low attachment dishes or flasks. Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (FBS) was used to induce the differentiation of GICs and to passage 293FT and PLAT-A human embryonic kidney cells in culture.
Dmem f12 serum free medium
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
DMEM/F12 serum-free medium is a cell culture medium formulation designed to support the growth and maintenance of a variety of cell types without the addition of serum. It provides a balanced and defined nutrient composition to facilitate cell proliferation and viability in a serum-free environment.
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Lab products found in correlation
Epidermal growth factor (egf), by Thermo Fisher Scientific (21 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (18 mentions)
Insulin, by Merck Group (18 mentions)
Bovine serum albumin (bsa), by Merck Group (13 mentions)
Epithelial growth factor (egf), by Thermo Fisher Scientific (8 mentions)
Basic fibroblast growth factor (bfgf), by Merck Group (8 mentions)
6 well ultra low cluster plates, by Corning (7 mentions)
Epidermal growth factor (egf), by Merck Group (7 mentions)
Insulin, by Thermo Fisher Scientific (6 mentions)
Fibroblast growth factor 2 (fgf2), by Thermo Fisher Scientific (5 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (5 mentions)
L glutamine, by Thermo Fisher Scientific (5 mentions)
B27 supplement, by Thermo Fisher Scientific (4 mentions)
Epidermal growth factor (egf), by BD (3 mentions)
Ultra low attachment culture dishes, by Corning (3 mentions)
Six well ultra low cluster plates, by Corning (3 mentions)
Inverted microscope, by Leica (2 mentions)
96 well clear ultra low attachment microplates, by Corning (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Ultra low adherent six well plates, by Corning (2 mentions)
54 protocols using dmem f12 serum free medium
1
Establishment and Characterization of Glioma Initiating Cells
2
Isolation and Culture of A549 Cancer Stem Cells
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Cancer stem cells were sorted from A549 cells or transfected-A549 cells by sphero-cyst medium [10 (link)], and the sorted cancer stem cells from A549 were named as A549-derived stem cells. The 3 × 104 cells were seeded in 6-well ultra-low cluster plates (Thermo Fisher Scientific). They were maintained in DMEM/F12 serum free medium (Thermo Fisher Scientific) containing epidermal growth factor (20 ng/mL), beta-fibroblast growth factor (20 ng/mL), insulin (4 μg/mL), and B27 (2%). All reagents were from Sigma. Finally, the number of spheres was counted under an inverted microscope (Leica, Oskar-Barnack-Straße, Germay) post 10 days incubation. The biomarkers of cancer stem cells SOX2, CD34, and CD133 were used to identify the cancer stem cells using qRT-PCR and Western blot.
The A549 cancer stem cells were isolated using magnetic bead kit, and 3 × 104 stem cells were seeded in six-well plates with ultra-low adhesion. They were cultured in sphere formation media for a week, and post another 20 days, the number of spheres was calculated under a microscope.
The A549 cancer stem cells were isolated using magnetic bead kit, and 3 × 104 stem cells were seeded in six-well plates with ultra-low adhesion. They were cultured in sphere formation media for a week, and post another 20 days, the number of spheres was calculated under a microscope.
3
Spheroid Formation Assay for HCT116 Cells
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HCT116 cells were seeded in six-well plates at a density of 1×105 cells per well, incubated at 37°C overnight, and transfected with miR-NC or miR-1291 or DCLK1-siRNA at a final concentration of 50 nM. After 24 h of transfection, single cells were reseeded in 96-Well Clear Ultra Low Attachment Microplates (Corning, Inc.) at a density of 1,000 cells per well. The cells were cultured in DMEM/F-12 serum-free medium (Thermo Fisher Scientific, Inc.) supplemented with 20 ng/ml epithelial growth factor, 10 ng/ml basic fibroblast growth factor-2 (PeproTech, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were cultured in a humidified incubator at 37°C and 5% CO2. Images were captured by a bright field light microscope (CKX53; Olympus Corporation) with Visualix camera (Visualix, Corporation). The number of spheres was counted manually on days 4 or 7 after reseeding.
4
Glioblastoma Cell Culture and Drug Treatment
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U87 (alias U87-MG) and A172 were purchased from ATCC and cultured in DMEM 4.5 g/L glucose (Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10% fetal bovine serum (Thermo Fisher Scientific). GSCs were isolated from patients’ surgical samples at the Institute of Neurosurgery, Catholic University of Rome as previously described [16 (link)] and grown in suspension in DMEM/F12 serum-free medium (Thermo Fisher Scientific) containing 2 mM L-glutamine, 0.6% glucose, 9.6 mg/mL putrescine, 6.3 ng/mL progesterone, 5.2 ng/mL sodium selenite, 0.025 mg/mL insulin, 0.1 mg/mL transferrin sodium salt (Sigma Aldrich, St Louis, MO, USA), EGF (20 ng/mL; Peprotech, London, UK), bFGF (10 ng/mL; Peprotech), and heparin (2 μg/mL; Sigma Aldrich) at 37 °C, 5% CO2. All cell lines were regularly checked to exclude mycoplasma contamination by Mycoalert Detection Kit (Lonza, Basel, Switzerland).
Regorafenib (Bay73-4506) was purchased from Selleckem (Houston, TX, USA) and resuspended in DMSO.
Regorafenib (Bay73-4506) was purchased from Selleckem (Houston, TX, USA) and resuspended in DMSO.
5
Sphere Culture Protocol for Cancer Research
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Six hundred stable cell lines were seeded in 6-well ultra-low cluster plates (Corning Incorporated, Corning, NY, USA) for 10 days. Spheres were cultured in DMEM/F12 serum-free medium (Thermo Fisher Scientific) supplemented with 2% B27 (Thermo Fisher Scientific), 20 ng/mL EGF, and 20 ng/mL bFGF (PeproTech, Offenbach, Germany), 5 μg/mL insulin, and 0.4% BSA (Sigma-Aldrich Co.).
6
Culturing Sphere-Forming Cells
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Five hundred cells were seeded in 6-well ultra-low cluster plates and 10 or 20 cells were seeded in 24-well ultra-low cluster plates for 10 days. Spheres were cultured in DMEM/F12 serum-free medium (Invitrogen, USA) supplemented with 2% B27 (Invitrogen, USA), 20 ng/ml EGF, 20 ng/ml bFGF, and 5 μg/ml insulin (PeproTech, USA).
7
Isolation and Characterization of Cancer Stem Cells
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The LSR II flow cytometer (BD Pharmingen, San Diego, CA, USA) was used to analyze and separate CSCs based on cell labeling and fluorescence-activated sorting. The activated ALDEFLUOR™ reagent and diethylaminobenzaldehyde purchased from Stemcell Technologies, Inc. (Vancouver, BC, Canada) were used to isolate aldehyde dehydrogenase (ALDH)+ cells (21 (link)–23 (link)). The ratios of ALDH1+ stem cells in different groups were analyzed. The ratios of aldH 1+ stem cells in different groups were analyzed. For sphere forming assay, CSCs (1000cells per/ well) were seeded in 6-well ultra-low cluster plates (Corning Inc., Corning, NY) for one week to obtain first generation after being given different treatment. Spheres were cultured in DMEM/F12 serum-free medium (Invitrogen) supplemented with 2% B27 (BD Biosciences, CA), 20ng/ml epidermal growth factor (EGF, Sigma), 20ng/ml basic fibroblast growth factor (bFGF, Sigma), 0.4% BSA and 4 μg/ml insulin (Sigma). The number of spheres was counted under a microscope.
8
HCC Spheroid Formation Protocol
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A total of 5 × 103 HCC cells were inoculated into six-well ultra-low adsorption plates (Corning, Painted Post, NY, United States) containing 2% B27 (BD PharMingen, Carlsbad, CA, United States), 20 ng/mL epidermal growth factor, 20 ng/mL fibroblast growth factor in DMEM/F12 serum-free medium (Invitrogen). The cells were cultured for 14 d and then imaged and counted under a light microscope.
9
Sphere Formation Assay for Stem Cells
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The same number of cells (1000) were seeded into ultralow attachment culture dishes and cultured in DMEM/F12 serum-free medium (Invitrogen) supplemented with 2% B27 (Thermo Fisher, USA), 1% N2 (Thermo Fisher, USA), 20 ng/ml epidermal growth factor (Sigma-Aldrich, USA) and 10 ng/ml basic fibroblast growth factor (BD, Franklin Lakes, NJ, USA), for 10 days. The cell spheres were observed and photographed by an inverted microscope, and the Image-Pro Plus software was used to measure the longest diameter of spheres by the contrast scale in the pictures.
10
Sphere Culture: Serum-Free Medium
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Cells (100 cells per well) were seeded in 6-well ultra-low cluster plates for 10 days. Spheres were cultured in DMEM/F12 serum-free medium (Invitrogen, San Diego, CA, USA) supplemented with 2% B27 (Invitrogen, San Diego, CA, USA), 20 ng/ml EGF, 20 ng/ml bFGF, and 5 μg/ml insulin (Invitrogen, San Diego, CA, USA).
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