This assay tests the ability of single cells to form tumorspheres, the in vitro surrogate of stem-like cells [42 (link)]. Cells (2 × 105 cells/well in 1.5 mL of medium) were distributed into an ultralow-attachment six-well plate. All tumorspheres were grown in DME-F12 medium (Gibco, Milan, Italy), supplemented with penicillin/streptomycin, L-glutamine, B27 supplement (1×, #17504-044, Life Technologies), bFGF (20 ng/mL, #13256029, Gibco), and hEGF (20 ng/mL, #10605-HNAE, Gibco), and allowed to grow for 7–10 days, or until the majority of spheres reached a diameter of 60 μm. Tumorspheres were treated with CM037 (10 μM) every 24 h. Tumorspheres were counted and then harvested followed by protein extraction or split for second and third tumorsphere generation and lysed for protein extraction [33 (link)].
B 27 supplement 1
Manufactured by Thermo Fisher Scientific
Sourced in United States
B-27™ Supplement (1×) is a cell culture supplement developed by Thermo Fisher Scientific. It is designed to support the growth and differentiation of neuronal and other central nervous system cell types in vitro. The supplement contains a proprietary, defined mixture of hormones, vitamins, antioxidants, and other components.
Automatically generated - may contain errors
Lab products found in correlation
L glutamine, by Thermo Fisher Scientific (3 mentions)
Heparin, by Merck Group (2 mentions)
Rhfgf basic, by R&D Systems (2 mentions)
Rhnodal, by R&D Systems (2 mentions)
Hepes buffer, by Merck Group (2 mentions)
Advanced dmem f12 medium, by Thermo Fisher Scientific (2 mentions)
Rhfgf 10, by R&D Systems (2 mentions)
Matrigel, by Corning (2 mentions)
Dme f12 medium, by Thermo Fisher Scientific (1 mentions)
Neurobasal medium, by Thermo Fisher Scientific (1 mentions)
B27 supplement, by Thermo Fisher Scientific (1 mentions)
Poly d lysine, by Merck Group (1 mentions)
Nerve growth factor (ngf), by Thermo Fisher Scientific (1 mentions)
L ascorbic acid, by Merck Group (1 mentions)
Ultra low attachment six well plate, by Corning (1 mentions)
Insulin, by Merck Group (1 mentions)
R spondin, by Thermo Fisher Scientific (1 mentions)
N acetylcystein, by Merck Group (1 mentions)
Advanced dmem f12, by Thermo Fisher Scientific (1 mentions)
Primocin, by Thermo Fisher Scientific (1 mentions)
11 protocols using b 27 supplement 1
1
Tumorsphere Formation Assay for Stem-like Cells
2
Isolation and Culture of Cortical Neurons
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Mice (c57/bl6 strain) were kept in a normal light/dark cycle (12 h light:12 h dark) and had free access to water and food. Cortical neurons were prepared from E15 female and male embryos using a previously described method [14 (link)]. Briefly, we euthanized the pregnant mice with a carbon dioxide (CO2) overdose. We isolated cortices under a stereomicroscope. After mechanical dissociation, cells were counted and plated on 12 mm coverslips coated with poly-D-lysine (Sigma, St. Louis, MO, USA) according to the density of 150–200 cells/mm2. The cells were grown in neuronal complete medium (Neurobasal 1×, B-27 supplement 1×, 0.5 mM L-glutamine, 10 μg/mL gentamicin; B-27 supplement and Neurobasal medium were purchased at Life Technologies, Carlsbad, CA, USA) at 37 °C and 5% CO2 until full maturation, i.e., 14 days in vitro (DIV).
3
Patient-Derived PDAC Culture Protocol
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All experiments with human material were performed in accordance with the Declaration of Helsinki and were approved by the ethics committee of the Medical Faculty of Heidelberg University (323/2004, Amendment 03). Informed consent was received from participants before study inclusion. Generation and cultivation of patient-derived PDAC cultures have been described previously (16 (link)). Cultures were subjected to SNP typing and Multiplex Cell Contamination Testing (Multiplexion). Cultures were grown in DMEM Advanced F12 medium supplemented with 0.6% (w/v) glucose, 2 mM L-glutamine, 2% B27 supplement (1×) (all Thermo Fisher Scientific), 12 μg/ml heparin and 5 mM HEPES buffer (both Sigma Aldrich), 10 ng/ml rhFGF basic, 20 ng/ml rhFGF-10, and 20 ng/ml rhNodal (all R&D Systems). Cytokines were renewed twice per week.
4
Neuronal Differentiation of Neural Progenitor Cells
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Neural progenitor cells (NPCs) were cultured with N-2 Supplement 1× (Thermo Fisher Scientific, Massachusetts, USA), B-27 Supplement 1× (Thermo Fisher Scientific, Massachusetts, USA), epidermal growth factor (EGF, 20 ng/mL, Peprotech, New Jersey, USA), basic fibroblast growth factor (bFGF, 20 ng/mL, Peprotech, New Jersey, USA) in DMEM/F12 media. NPCs were seeded in 24-well cell culture plates coated with Matrigel (354234, 1/200, Corning, New York, USA). Cells were cultured in an incubator at 5% CO₂ and 37 °C. Cells were subcultured every 4–5 days, and the medium was changed daily.
For neuronal differentiation, NPCs were changed with DMEM/F12 medium containing N-2 Supplement 1×, B-27 Supplement 1×, brain-derived neurotrophic factor (BDNF, 10 ng/mL, Peprotech, New Jersey, USA), glial cell-derived neurotrophic factor (GDNF), 10 ng/mL, Peprotech, New Jersey, USA), nerve growth factor (NGF, 10 ng/mL, Peprotech, New Jersey, USA), and 0.1 mM L-Ascorbic acid (Sigma Aldrich, Missouri, USA) with 5 × 107 particles/mL of dEVs. The medium was changed daily, and the cells were cultured for 7 days.
For neuronal differentiation, NPCs were changed with DMEM/F12 medium containing N-2 Supplement 1×, B-27 Supplement 1×, brain-derived neurotrophic factor (BDNF, 10 ng/mL, Peprotech, New Jersey, USA), glial cell-derived neurotrophic factor (GDNF), 10 ng/mL, Peprotech, New Jersey, USA), nerve growth factor (NGF, 10 ng/mL, Peprotech, New Jersey, USA), and 0.1 mM L-Ascorbic acid (Sigma Aldrich, Missouri, USA) with 5 × 107 particles/mL of dEVs. The medium was changed daily, and the cells were cultured for 7 days.
5
Generation and Characterization of Patient-Derived Pancreatic Cancer Cultures
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Patient-derived pancreatic cancer cultures PC9 and PC18 were generated and cultivated as described previously [22 ]. Cultures were subjected to SNP typing and Multiplex Cell Contamination Testing (Multiplexion, Heidelberg, Germany). PC9 and PC18 cultures were grown in DMEM Advanced F12 medium supplemented with 0.6% (w/v) glucose, 2 mM L-glutamine, 2% B27 supplement (1×) (all Thermo Fisher Scientific), 12 μg/mL heparin and 5 mM HEPES buffer (both Sigma Aldrich), referred to as CSCN medium. Cytokines, i.e., 10 ng/mL rhFGF-basic, 20 ng/mL rhFGF-10, and 20 ng/mL rhNodal (all R&D Systems) were added to the cultures and renewed twice a week.
6
Sphere Formation Assay for Gastric Cancer
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Tumorspere cultures were cultured in ultralow attachment six-well plate (Corning, Lowell, MA, USA) in suspention (500 cells/mL) in serum-free DMEM/F12 media, supplemented with 20 ng/mL epidermal growth factor (FGF, Sigma-Aldrich, Shanghai, China), 4 μg/mL insulin (Sigma-Aldrich, Shanghai, China), B27 supplement (1×, Invitrogen, Carlsbad, CA, USA), 1% of penicillin-streptomycin in humidified incubator at 37 °C in 5% CO2. Tumor sphere formation was tested by placing gastric cancer cells in presence or absence of genistein under the conditions mentioned above. After 7 day incubation, the solid spheres were photographed and counted. The sphere size varies greatly from less than 50 μM to around 250 μM. With tumorspheres, the cells appear fused together and it is difficult to distinguish them as individual cells. With aggregated cells that were not counted, you can still see individual cells attached to one another.
7
Protocol for Intestinal Organoid Generation
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For generating intestinal organoids, crypts from C57BL6J mice were used. Mice were anesthetized using CO2 and crypts were isolated from the small intestine by incubation of tissue for 25 min at 4 °C in crypt isolation buffer (CIB, PBSO containing 0.5 M EDTA). Isolated crypts were counted and a total of 500 crypts were plated with 25 µL Matrigel (Corning B.v., Amsterdam, Netherlands) in a 48-well plate. After polymerization of Matrigel, 300 µL of crypt culture medium (CCM) consisting of advanced DMEM/F12 (ThermoFisher Scientific, Karlsruhe, Germany) was supplemented with 100 ng/µL Noggin (PeproTech, East Windsor, NJ, USA), 1 µg/mL R-Spondin (PeproTech, East Windsor, NJ, USA), B-27™ supplement 1× (Invitrogen, Carlsbad, CA, USA), 1 mM N-Acetylcystein (Sigma-Aldrich, Darmstadt, Germany), 0.1 mg/mL Primocin (Invitrogen, Carlsbad, CA, USA) and 50 ng/mL recombinant mouse epidermal growth factor (rm EGF) (Immunotools, Friesoythe, Germany). The resulting organoids were cultured and splitted for a minimum of seven days according to Sato et al. 2009 [33 (link)].
8
EGFR siRNA Knockdown Efficiency in DRG Cells
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EGFR siRNA (5′-GAUGGAGUCAGCAAGUGUATT-3′; 5′-UACACUUGCUGACUCCAUCTT-3′) and negative control (NC) siRNA (5′-UUCUCCGAACGUGUCACGUTT-3′; 5′-ACGUGACACGUUCGGAGAATT-3′) were designed and synthesized by Genepharma (Shanghai, China). The DRGs from three- to four-week-old SD rats were harvested and cultured for examining the knockdown efficiency of EGFR siRNA. All harvested DRGs were then digested with 0.25% trypsin solution without EDTA (Beyotime Biotechnology, Shanghai, China). Following trituration and centrifugation, dissociated cells were resuspended and cultured in cold Neurobasal™-A Medium (Gibco/ThermoFisher Scientific, Waltham, MA) with 10% fetal bovine serum (JR Scientific, Woodland, CA), B-27™ Supplement (1×) (Gibco/ThermoFisher Scientific), 100 units per ml penicillin, and 100 µg per ml streptomycin (Beyotime Biotechnology) in a six-well plate precoated with 50 µg per ml poly-D-lysine (Beyotime Biotechnology). The cultured cells were incubated in an incubator with 95% O2, 5% CO2, and at 37° 7. After 24 h incubation, EGFR siRNA (250 pmol) or equivalent NC siRNA was delivered by Lipo6000 transfection reagent (Beyotime Biotechnology) into cultured cells in six well plates. Two days later, the cultured cells were harvested into radio immunoprecipitation assay (RIPA) lysis buffer for western blotting.
9
Culturing and Quantifying WT and DOXR Spheroids
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A total of 1 × 105 WT or DOXR cells were cultured on ultra-low-attachment 6-well plates (Corning, USA) with DMEM/F12 (Gibco) medium containing 2 ng/mL epidermal growth factor (EGF) (PeproTech, USA), 2 ng/mL FGF (PeproTech), B-27 supplement (1×) (Gibco), and 1% antibiotics (Gibco). After 8 days, the number of WT and DOXR spheroids with a diameter over 100 μm was counted.
10
Differentiation of iPSCs to Innervated Skeletal Muscle
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The differentiation to innervated skeletal muscle was performed following the protocol previously described by Mazaleyrat et al., 2020 [29 (link)]. After reaching a high confluence, iPSCs growing in mTeSR™1 were manually picked and seeded on hESC-qualified Matrigel-coated plates. For the differentiation of iPSCs to skeletal muscle cells the basal medium for the maintenance of the cells is composed by Neurobasal™ (Gibco, Waltham, MA, USA; 21103-049), supplemented with GlutaMAX™ 1× (Invitrogen, Waltham, MA, USA; 35050-038), Penicillin-Streptomycin 1× (Gibco, Waltham, MA, USA; 15140122), non-essential amino acids 1× (Merck, Darmstadt, Germany; M7145), N-2 supplement 1× (Gibco, Waltham, MA, USA; 17502048) and B-27™ supplement 1× (Gibco, Waltham, MA, USA; 17504044). To induce myogenic and motor neuron differentiation subsequent medium daily changes were performed with the basal medium supplemented with the compounds shown in Table 3 [49 (link)]. The iPSC lines which were differentiated towards skeletal muscle cells in this study were C10 [27 (link)] and AG09G (kindly gifted by Dr Magdinier’s group) as control lines and MA1 [28 (link)] and MA4 as McArdle lines.
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