Cortical neurons were isolated and cultivated as described in our previous studies [59 (link), 60 (link)]. Primary cortical neurons were derived from embryonic 17th C57BL/6 mice. After euthanizing pregnant mice, the mice were sterilised in an ice bath containing 75% alcohol for 5 min. Then, the abdominal cavity was opened in a sterile environment in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, 31053028). Under a stereomicroscope (Olympus, Tokyo, Japan), the fetal cerebral cortical tissue was dissected and cut into small pieces (1 mm3), followed by incubation in 2 mg/ml papain (Worthington, LS03126) and 20 units/ml DNase for 15 min at 37 °C (Sigma-Aldrich, P4762). Subsequently, the cells were dispersed in DMEM containing 10% fetal bovine serum (Gibco, 10099-141). The cell density was adjusted to 50,000 cells/cm2 by counting, and the cells were plated in a Petri dish coated with 0.01% poly-D-lysine (Sigma-Aldrich, P4707) in a 37 °C incubator under an atmosphere with 5% CO2. After 4 h, the previous medium was replaced with neurobasal medium (Gibco, 21103049) containing 1% B27 additive (Thermo Scientific, 17504044), 2 mM L-glutamine (Gibco, 25030081) and 1% penicillin/streptomycin (Thermo Scientific, 15140148) at 37 °C under an atmosphere with 5% CO2 for cultivation in an incubator, with the culture medium replaced every three days.
B27 additive
Manufactured by Thermo Fisher Scientific
Sourced in United States
B27 Additive is a serum-free supplement used to support the growth and differentiation of neurons and other neural cell types in cell culture. It is a defined, animal component-free mixture of vitamins, antioxidants, and other nutrients that promotes neuronal survival and function.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Insulin, by Merck Group (3 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (3 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Sb202190, by Merck Group (2 mentions)
Epidermal growth factor (egf), by Merck Group (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Nicotinamide, by Thermo Fisher Scientific (2 mentions)
A83 01, by Bio-Techne (2 mentions)
Hepes, by Merck Group (2 mentions)
Y 27632, by Selleck Chemicals (2 mentions)
Noggin, by Thermo Fisher Scientific (2 mentions)
Fibroblast growth factor 2 (fgf2), by Thermo Fisher Scientific (2 mentions)
Fgf10, by Thermo Fisher Scientific (2 mentions)
N acetyl l cysteine, by Merck Group (2 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
Pbs buffer, by Thermo Fisher Scientific (2 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (2 mentions)
Neurobasal medium, by Thermo Fisher Scientific (2 mentions)
12 protocols using b27 additive
1
Isolation and Culture of Cortical Neurons
2
Endoderm Differentiation of t-PSC Cells
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t-PSC cells were mechanically dissociated and plated on Matrigel coated 96-well plates, and treated with CWP232228 (0.1μM) or I-CBP112 (0.25μM) in mTeSR-1 media (STEMCELL Technologies) for 48 hours. Control (plain media and DMSO) and treated cells were incubated in endoderm differentiation media (RPMI 1640 supplemented with B-27 additive (Thermo Fisher), 100ng/ml of Activin A (PeproTech), 3uM of CHIR99021 (Tocris), and 100ng/ml of HGF (Peprotech)) over 6 days. Then, cells were fixed and permeabilized (Fixation/Permeabilization Solution Kit, BD Bioscience) and immunostained as described above, using anti-OCT4 and anti-FOXA2 primary antibodies (key resources table ). Fluorescence images were acquired and analyzed using an ImageXpress Pico High-Content imaging system (Molecular Devices), as described in the immunofluorescence analysis section.
3
Neuronal Cell Culture and Analysis
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The following materials were used for the procedures described in this article: DMEM high Glucose Medium (Thermo company, USA); FBS (Gibco, USA); B27 Additive (Invitrogen, USA); Neurobasal-A culture medium (Thermo Basal, USA); the MTT (Invitrogen, Waltham, USA); DMSO (Sigma, USA); Polylysine (Sigma, USA); 0.25% trypsin (Gibco, USA); PBS buffer (Thermo, USA); Rabbit anti-mouse NeuN monoclonal antibodies, Goat rabbit antigens (Wuhan Bioengineering Co., Ltd.); Goat anti-catalase antibodies (American R&D Company); Goat anti-protein kinase A antibodies (American R&D company); Rabbit anti-goat IgG (Jiangsukaiji Biotechnology Co., Ltd.); Cisphenolate ELISA kit (Shanghai Meixuan Biotechnology Co., Ltd.); Magnetic stimulator (model MagPro R3,Medtronic company, Denmark).
4
Organoid Culture Media Composition
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Clevers media is comprised of Advanced DMEM/F12 with the following additives: 50 ng/mL EGF (Sigma), 5% v/v R-spondin 1, 10% v/v Noggin, 10 ng/mL FGF10 (Peprotech), 1 ng/mL FGF2 (Peprotech), 10 nM Nicotinamide (Acros), 0.5 µM A83-01 (Tocris), 10 µM SB202190 (Sigma Aldrich), 10 µM Y-27632 (Selleck Chemical), 1X B27 Additive (Invitrogen), 1.25 mM N-Acetyl-L-cysteine (Sigma-Aldrich), 2 nM Glutamax (Invitrogen), 10 mM HEPES (Sigma Aldrich), 1:100 v/v Primocin (Invitrogen), 10% Foetal Bovine Serum as previously described (37 (link)). The enriched cells were cultured in Clevers media under hypoxic conditions (1–2% O2).
5
Pluripotent Stem Cell Characterization
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We obtained Roswell Park Memorial Institute (RPMI) 1640 medium, TRizol total RNA extraction reagent, and diethyl pyrocarbonate (DEPC) water (TIANGEN BIOTECH, Beijing, China); Trans2K Plus II DNA Marker and FBS (Qiagen, Dusseldorf, Germany); goat anti-mouse secondary antibody, Oct4 antibody, Sox2 antibody, CD44 antibody, E-cadherin antibody, and β-actin antibody (Abcam, Cambridge, USA); PrimeScript RT Enzyme Mix I (x 200) and SYBR Green 820A (Takara, Tokyo, Japan); and Oct4 primer, Sox2 primer, CD44 primer, E-cadherin primer, KLF4 primer, c-Myc primer, B27 additive, human epidermal growth factor (EGF), and human basic fibroblast growth factor (bFGF; Invitrogen, Carlsbad, USA).
6
3D ex-vivo CTC Expansion Protocol
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Isolated CTCs were cultured in the following formats: 96 well standard microplates, 96F non adherent microplates, spheroid microplates (Thermo Scientific, USA) and specialized microwell formats with a culture medium containing Advanced DMEM/F12 with the following additives: 50 ng/mL EGF (Sigma), 5% v/v R-spondin 1, 10% v/v Noggin, 10 ng/mL FGF10 (Peprotech), 1 ng/ml FGF2 (Peprotech), 10 nM Nicotinamide (Acros), 0.5 μM A83–01 (Tocris), 10 μM SB202190 (Sigma Aldrich), 10 μM Y-27632 (Selleck Chemical), 1X B27 Additive (Invitrogen), 1.25 mM N-Acetyl-L-cysteine (Sigma-Aldrich), 2 nM Glutamax (Invitrogen), 10 mM HEPES (Sigma Aldrich), 1:100 v/v Primocin (Invivogen). For 3D ex-vivo expansion, Happy Cell® (Biocroi Ltd, Dublin, Ireland) hydrogel was used in combination with the above mentioned cocktail to provide the environment for the CTCs. To perform spheroid staining, an inactivation buffer was added to the culture for 1 hour at 37oC to collapse the Happy Cell® matrix. Cultures were incubated under hypoxic conditions (2% O2, 5% CO2) and imaged weekly starting on day 7.
7
Clonogenic and Sphere-Forming Assays
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In the clonogenic assay, 250 cells were seeded in 100-mm² dishes. After 15 days, wells were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet and counted.
For the sphere-forming capacity, a ranging from 1024 cells to 1 cell were cultured in a sphere medium consisting of phenol red-free DMEM-F12 (Gibco), 0.4% bovine serum albumin (Sigma-Aldrich, St Quentin Fallavier, France), 10 mL of B27 additive (Invitrogen, Illkirch, France), 5 mg/mL of insulin (Sigma-Aldrich), 4 µg/mL of heparin and 20 ng/mL of epidermal growth factor (EGF) and fibroblast growth factor (FGF) (Biotechne, Abingdon, OX, United Kingdom). Cells were seeded in 96-well low-adhesion plates. The number of spheres per well were counted 4 days later.
For the sphere-forming capacity, a ranging from 1024 cells to 1 cell were cultured in a sphere medium consisting of phenol red-free DMEM-F12 (Gibco), 0.4% bovine serum albumin (Sigma-Aldrich, St Quentin Fallavier, France), 10 mL of B27 additive (Invitrogen, Illkirch, France), 5 mg/mL of insulin (Sigma-Aldrich), 4 µg/mL of heparin and 20 ng/mL of epidermal growth factor (EGF) and fibroblast growth factor (FGF) (Biotechne, Abingdon, OX, United Kingdom). Cells were seeded in 96-well low-adhesion plates. The number of spheres per well were counted 4 days later.
8
Ovarian Cancer Cell Culture Protocol
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The human epithelial ovarian cancer cell line, A2780, was purchased from Sigma. The human ovarian adenocarcinoma cell line, 3AO, was obtained from Women's Hospital, School of Medicine, Zhejiang University, which was tested and authenticated. 3AO and A2780 cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (BI, Kibbutz Beit-Haemek, Israel), supplemented with 10% fetal bovine serum (FBS) (Invitrogen, New York, NY, USA) at 37°C and 5% CO2. The adherent cells were cultured in serum-free medium (SFM) composed of 10uL/mL B27 additive (Life Technologies, Carlsbad, CA, USA), 10 ng/mL, 1 mg/mL insulin (Sigma-Aldrich, Burlington, MA, USA), basic fibroblast growth factor, 20 ng/mL epidermal growth factor (Pepro-Tech, Rocky Hill, CT, USA), Dulbecco's modified Eagle's medium (DMEM/F12) (BI), to form spheroids after plating 5×104 cells per well in ultra-low attachment 6-well culture plates (Corning, New York, NY, USA). The culture medium will be renewed every two or three days. After plating 400 or 600 cells per well in ultra-low attachment 96-well culture plates (Corning), A2780 or 3AO cells were cultured in SFM at 37°C in 5% CO2 for 7 days. The culture medium will be renewed every two or three days. The spheroids were cultured in RPMI-1640 medium with 10% FBS after plating in 6 Nunclon Delta plates (Thermo Scientific, Suzhou, China) to re-differentiate.
9
Culturing Ovarian Cancer Stem Cells
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Ovarian adenocarcinoma cell line 3AO was obtained from the Women’s Hospital School of Medicine, Zhejiang University. Cells were maintained in RPMI 1640 medium (Cellgro, Virginia) supplemented with 10% fetal bovine serum (FBS, Gibco). Ovarian CSC-LCs, the tumor spheroids, were cultured at a density of 50,000 cells/mL in serum-free DMEM/F12 medium (Cellgro, Virginia) composed of 10 ng/mL basic fibroblast growth factor and 20 ng/mL epidermal growth factor (PeproTech Inc, Rocky Hill, NJ), 1 mg/mL insulin (Sigma-Aldrich St Louis, MO), and 10 μL/mL B27 additive (Life Technologies, Carlsbad, CA) on ultralow attachment culture dishes (Corning, NY). All cells were maintained in a humidified atmosphere 5% CO2 at 37°C.
10
Preparation of Mouse Striatal Neuron Cultures
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Mouse striatal neuron cultures were prepared as previously described [23] (link), [24] (link). Briefly, striata from E15-E16 ICR mice were dissected, minced and enzymatically dissociated with trypsin (2.5 mg/ml) and DNase (0.015 mg/ml) in neurobasal medium (Gibco) for 30 min at 37°C. Tissue was resuspended in neurobasal medium supplemented with B-27 additives (Gibco), L-glutamine (0.5 mM; Gibco) and glutamate (25 µM; Sigma-Aldrich), triturated and filtered twice through 70 µm nylon mesh pore filters. Neurons were plated and maintained in supplemented neurobasal medium. Culture purity was determined by immunocytochemistry using anti-MAP-2 antibody (Abcam, Cambridge, MA; ab32454) and found to be >80% neurons.
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