PDA cells were resuspended in tumorsphere DMEM/F12 medium (Thermo Fisher Scientific Inc., Dreieich, Germany) supplemented with B27 supplement (50×) (Thermo Fisher), 20 ng/mL rEGF (Thermo Fisher), 20 ng/mL bFGF (Thermo Fisher), 5 μg/mL I nsulin (Sigma, Deisenhoffen, Germany) and 5 mL Glutamax (Thermo Fisher). To avoid false positives as a result of cell aggregation, 1000 cells in 50 μL tumorsphere medium mixed with 50 μL matrigel were cultured per well of a 24-well plate. The mixture was allowed to polymerize for 1 h at 37 °C and 5% CO2. Subsequently, 1 mL of the tumorsphere medium was added per well, either alone or supplemented with 50 nM mirVana™ mimics or 50 μM quercetin. Seventy-two hours later, the spheres were collected by gentle centrifugation, dissociated into single cells, and cultured for the formation of next generation spheres. Tumor spheres were counted using an inverted microscope (Nikon Eclipse TS100; Nikon GmbH, Düsseldorf, Germany). To quantify cell numbers, the tumor spheres were collected and disassociated with Trypsin-EDTA (Thermo Fisher) to make a single cell suspension. The viable cells were then counted using the Z™ Series COULTER COUNTER® Cell and Particle Counter (Beckman Coulter, Indianapolis, USA).
B27 supplement 50
Manufactured by Thermo Fisher Scientific
Sourced in United States
B27 supplement (50x) is a concentrated cell culture medium supplement formulated to support the growth and differentiation of neurons and other cell types. It provides a variety of components including antioxidants, insulin, transferrin, and other factors to promote cell viability and function.
Automatically generated - may contain errors
Lab products found in correlation
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (4 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Glutamax, by Thermo Fisher Scientific (3 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (3 mentions)
Dmem f12, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
F 12 dmem, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Merck Group (2 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (2 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (2 mentions)
N2 supplement 100, by Thermo Fisher Scientific (2 mentions)
Accutase, by Thermo Fisher Scientific (2 mentions)
Epidermal growth factor (egf), by Merck Group (2 mentions)
Neurobasal medium, by Thermo Fisher Scientific (2 mentions)
Insulin, by Merck Group (1 mentions)
Eclipse ts100, by Nikon (1 mentions)
Huvecs, by National Collection of Authenticated Cell Cultures (1 mentions)
P27kip1, by Proteintech (1 mentions)
Dulbecco s modified eagle s medium dmem f 12 medium, by Thermo Fisher Scientific (1 mentions)
Mccoy s 5a medium, by Thermo Fisher Scientific (1 mentions)
16 protocols using b27 supplement 50
1
PDA Cell Sphere Formation Assay
2
Neurosphere Formation and Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For neurosphere formation, 4 × 105 cells were cultured in 35-mm low-attachment culture dishes (Falcon) for 7 days in neurosphere-forming medium composed of Nutrient Mixture F-12 (DMEM/F12) (GIBCO) supplied with B-27 Supplement 50× (ThermoFisher Scientific), bFGF 20 ng/mL and EGF 10 ng/mL (Peprotech) [16 (link)]. The formed spheres were then transferred to 24-well plates and imaged with an inverted microscope (EVOS Digital Microscopes). Sphere number and size were measured using ImageJ software (http://rsb.info.nih.gov/ij/ ). Spheres were then stained with Nestin (1:100, DSHB) and β-III tubulin as described above (Immunofluorescence section).
3
Culturing GSC23 and HUVECs for Cell Research
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The GSC line GSC23, of human origin, was grown in a culture medium composed of Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12), enhanced with 20 ng/ml basic fibroblast growth factor (bFGF), 20 ng/ml epidermal growth factor and B27 supplement (50×) (all obtained from Gibco, Carlsbad, CA, USA). Human umbilical vein endothelial cells (HUVECs, Passages 3–4) were procured from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and maintained in DMEM enriched with 10% fetal bovine serum (FBS) (sourced from Gibco). Both cell types were grown in an environment of 37°C and 5% CO2. The culture medium was refreshed every 48–72 h.
4
Characterization of Stem Cell Marker Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
McCoy's 5A medium, Dulbecco's modified Eagle's medium (DMEM)/F12 medium, fetal bovine serum and B27 supplement (50×) were obtained from Gibco; Thermo Fisher Scientific, Inc. Penicillin G, streptomycin and Heparin Na salt were purchased from Sigma-Aldrich; Merck KGaA. Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were purchased from PeproTech, Inc. The primary antibodies [octamer-binding transcription factor 4 (Oct4) (cat. no. sc-5279), Sox2 (cat. no. sc-365823) and Nanog (cat. no. sc-293121)] used for immunofluoresence assay were purchased from Santa Cruz Biotechnology, Inc. The primary antibodies used for western blot analysis were purchased from the following companies: Cell Signaling Technology, Inc. [Oct4 (cat. no. 2750), Sox2 (cat. no. 3579), Nanog (cat. no. 4903), β-catenin (cat. no. 8480), transcription factor 4 (TCF4) (cat. no. 2569), c-Myc (cat. no. 18583) and survivin (cat. no. 2808)], Elabscience® [Akt (cat. no. E-AB-30471), phospho-Akt-473 (cat. no. E-AB-21135), glycogen synthase kinase (GSK)-3β (cat. no. E-AB-20885) ], American Research Products, Inc. [CDK2 (cat. no. A2439), cyclin D1 (cat. no. 10-M1033) and p21Cip1/WAF1 (cat. no. 24-1026-MSM3)] and ProteinTech Group, Inc. [(p27Kip1 (cat. no. 25614-1-AP), cyclin A2 (cat. no. 27242-1-AP), GAPDH (cat. no. 60004-1-Ig) and β-actin (cat. no. 20536-1-AP)].
5
Culturing patient-derived GBM cell line
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
N15-0385 patient-derived GBM cell line was established by the GlioTex team (GBM and Experimental Therapeutics) in the Institut du Cerveau et de la Moelle epiniere (ICM) laboratory. Cells were cultured in DMEM-F12 medium (Gibco Life Technologies) containing B27 supplement 50× (2%, Gibco Life Technologies), human bFGF (20 ng ml−1, Peprotech), human EGF (20 ng ml−1, Peprotech), penicillin (100 U ml−1, Sigma-Aldrich), streptomycin (100 mg ml−1, Sigma-Aldrich), heparin (5 μg ml−1, Alfa Aesar) and maintained in a humidified incubator at 37°C and 5% CO2. Cells were routinely tested for mycoplasma infection. For cell growth curve, cells were dissociated using Accutase (Thermo Fisher Scientific), and seeded into six-well cell culture plates ( ). Cells were counted each day using a Countess automated cell counter (Thermo Fisher Scientific, Waltham, MA, USA) and growth curves determined from live cell numbers over a 144 h period.
6
Serum-free 3D Sphere Culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
EOC cells were seeded in ultra-low attachment 6 well plates (Corning, Kennebunk ME, USA) and cultured in serum free DMEM/F12 supplemented with 2% B27 Supplement (50×) (Gibco, Grand Island NY, USA), basic fibroblast growth factor (bFGF, Sigma-Aldrich, Saint Louis MO, USA) and epidermal growth factor (EGF, Sigma-Aldrich) with the final concentration of 20ng/μL at the temperature of 37 °C in a 5% CO2 incubator. Spheroids were recorded and counted under microscope after 2-4 weeks.
7
Isolation and Culture of Hippocampal Neurons
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All animal experiments
were performed under the guidelines of Animals (Control of Experiments)
Ordinance, approved by the Committee on the Use of Human and Animal
Subjects in Teaching and Research of the Hong Kong Baptist University
and conformed to The Principles of Laboratory Animal Care (NIH publication
no. 86-23, revised 1985). Sprague–Dawley rats were obtained
from the Chinese University of Hong Kong. Hippocampus neuronal cells
were isolated from 1-day-old Sprague–Dawley rats. Briefly,
the brains were placed in cold Hank’s balanced salt solution
(HBSS; Gibco), and the hippocampus cells were extracted. The cells
were resuspended in DMEM/F-12 medium (Gibco) with 10% fetal bovine
serum (Gibco) and papain solution (Sigma-Aldrich) in 2 μg/mL
was added into the system. The mixture was incubated at 37 °C
for 20 min under gentle shaking. The solution was purified by a 0.4
μm nylon filter (BD falcon), and the filtrate was further centrifuged
at 1000 rpm for 5 min. The pellet containing the dissociated neurons
was resuspended in neurobasal medium (Gibco) with supplements 2% B-27
(B-27 Supplement 50; Gibco), 0.2% penicillin (PSN; Gibco), and 0.25%
glutamax (GlutaMAX-I 100X; Gibco). The centrifugation steps were repeated
twice. The cells were plated into a poly-d -lysine (Sigma-Aldrich)-coated
plate and incubated in a humidified incubator at 37 °C with 5%
CO2 for 7 days before treatment.
were performed under the guidelines of Animals (Control of Experiments)
Ordinance, approved by the Committee on the Use of Human and Animal
Subjects in Teaching and Research of the Hong Kong Baptist University
and conformed to The Principles of Laboratory Animal Care (NIH publication
no. 86-23, revised 1985). Sprague–Dawley rats were obtained
from the Chinese University of Hong Kong. Hippocampus neuronal cells
were isolated from 1-day-old Sprague–Dawley rats. Briefly,
the brains were placed in cold Hank’s balanced salt solution
(HBSS; Gibco), and the hippocampus cells were extracted. The cells
were resuspended in DMEM/F-12 medium (Gibco) with 10% fetal bovine
serum (Gibco) and papain solution (Sigma-Aldrich) in 2 μg/mL
was added into the system. The mixture was incubated at 37 °C
for 20 min under gentle shaking. The solution was purified by a 0.4
μm nylon filter (BD falcon), and the filtrate was further centrifuged
at 1000 rpm for 5 min. The pellet containing the dissociated neurons
was resuspended in neurobasal medium (Gibco) with supplements 2% B-27
(B-27 Supplement 50; Gibco), 0.2% penicillin (PSN; Gibco), and 0.25%
glutamax (GlutaMAX-I 100X; Gibco). The centrifugation steps were repeated
twice. The cells were plated into a poly-
plate and incubated in a humidified incubator at 37 °C with 5%
CO2 for 7 days before treatment.
8
Differentiation of iPSC-derived Motoneurons
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The differentiation of motoneurons derived from iPSCs was performed as previously described [21 (link)]. Briefly, iPSCs were dissociated with dispase (Gibco, 1 mg/mL) or accutase (Stem cell) and cultured in MN induction medium, including Neurobasal medium (Invitrogen), DMEM/F12 (Hyclone) at 1:1, 0.5 × B27 supplement (50×) (Gibco), 0.5 × N2 supplement (100×) (Gibco), and 1 × GlutaMAX (100×) (Gibco). Different combinations of DMH1 (Selleck Chemicals, Houston, TX, USA), retinoic acid (RA) (Sigma-Aldrich), CHIR99021 (Selleck), SB431542 (Selleck), valproic acid (Sigma-Aldrich), purmorphamine (Pur) (Selleck), and DAPT (Selleck) were added to the medium at different stages (Figure 2 A).
9
Mammosphere-Formation Assay for Stem Cell Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The mammosphere‐formation assay has been adapted to quantify stem cell activity and self‐renewal. According the detailed protocol recommended by Shaw et al,18 the stable transfection cell lines (shFLI‐1 NC and 1# of MDA‐MB‐231 cells) were seeded into a 6‐well plate with ultralow attachment surface (#3471, Corning Inc., Corning, NY, USA) and cultured in DMEM/F12 (Thermo Scientific HyClone, Beijing, China) supplemented with 10 ng/mL recombinant human epidermal growth factor (EGF) (Cat. no. AF‐100‐15; Peprotech, Rochy Hill, NJ, USA), 10 ng/mL recombinant human basic fibroblast growth factor (bFGF) (Cat. no. 100‐18B; Peprotech), 2% B27™ supplement 50 × (LOT. 1860141; Gibco, Gaithersburg, MD, USA). Seven days after incubation, the plates were gently moved and observed to count the number of mammospheres formed. The mammospheres (magnification, 400 × ) of diameter >50 μm were judged as an effective mammosphere.
10
Nitidine Chloride Inhibition of Breast Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Breast cancer cell lines MCF-7 and MDA-MB-468 were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA), and were routinely cultured in DMEM medium (GIBCO Laboratories, Grand Island, NY, USA) supplemented with 10 % FBS (Haoyang biological manufacture, Tianjin, China), 100 U/ml penicillin and 100 mg/ml streptomycin. Nitidine chloride was obtained from Tauto Biotech (Shanghai, China) and dissolved in dimethyl sulfoxide (DMSO). Epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and bovine insulin were obtained from Sigma Aldrich (St. Louis, MO, USA). B27 supplement (50×) was purchased from Gibco. For NC treatment, stock solution (5 mM in DMSO) was added into culture medium to achieve appropriate concentration (1 or 2.5 μM), and then incubated with cells, whereas DMSO solution without NC was used as a control.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!