Embryoid body (EB) formation was performed by plating single cells in AggreWell800 (Stem Cell Technologies, 34811, Vancouver, Canada) in medium containing Knockout DMEM F-12 (Gibco,12660-012, Grand Island, NY, USA), 20% Knockout Serum (Gibco, 10828-028, Grand Island, NY, USA), non-essential amino acids-1x (Gibco, 11140-050) and Glutamax-1x (Gibco, 35050-061). Medium was changed 48 hours later and thereafter till day 7 in an every-other-day mode. On day 7, EB spheres were collected and plated onto plates coated with 0.1% Gelatin (Millipore, ES-006-B) and medium containing Dulbecco’s modified Eagle’s medium (DMEM) (Gibco 11965-092), 20% fetal bovine serum (FBS) (Gibco, SH30071), non-essential amino acids-1x (Gibco, 11140-050) and Glutamax-1x (Gibco, 35050-061). Medium was changed every other day for 7 days. On day 7 post plating, EBs were fixed with 4% paraformaldehyde (Santa Cruz, SC-281692, Dallas, TX, USA), and stained for detection of cells of the three germ layers with antibodies for the following antigens: SOX17 (R&D Systems, AF1924, Minneapolis, MN, USA) for endoderm, PAX6 (BioLegend, PRB-278P, San Diego, CA, USA) for ectoderm and SMA (Millipore, CBL171) for mesoderm.
Non essential amino acids 1x
Manufactured by Thermo Fisher Scientific
Sourced in United States, Denmark
Non-essential amino acids 1X is a mixture of amino acids that are not required for human growth and development. It provides a balanced source of these compounds for cell culture and other research applications.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780, by Zeiss (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
L glutamine, by Thermo Fisher Scientific (3 mentions)
Penicillin, by GE Healthcare (2 mentions)
Streptomycin, by GE Healthcare (2 mentions)
Rpmi 1640 with glutamax, by Thermo Fisher Scientific (2 mentions)
β mercaptoethanol, by Merck Group (2 mentions)
Accutase, by Thermo Fisher Scientific (2 mentions)
Hepes, by Thermo Fisher Scientific (2 mentions)
Glutamax 1x, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
75 cm2 flask, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Lonza (2 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (2 mentions)
Heat inactivated fetal calf serum, by Thermo Fisher Scientific (1 mentions)
Na pyruvate, by Thermo Fisher Scientific (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
13 protocols using non essential amino acids 1x
1
Differentiation of Stem Cells into Germ Layers
2
Culturing Human Basophils and Mouse Splenocytes
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Human basophils and mouse splenocytes were cultured in culture medium (RPMI 1640 with Glutamax and 20 mM HEPES, 1 mM Na-pyruvate, non-essential amino acids 1X (all from Life Technologies, Saint-Aubin, France)), 100 µg/mL streptomycin and 100 U/mL penicillin (GE Healthcare, Vélizy, France) and 37.5 μM β-mercaptoethanol (Sigma-Aldrich, MO) supplemented with 20% heat-inactivated fetal calf serum (Life Technologies) at 37 °C and 5% CO2.
3
Mouse Embryonic Explant Culture
4
Splenocyte Stimulation and Analysis
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Mouse splenocytes were harvested as described above and resuspended at 5 million cells/mL in culture medium (RPMI 1640 with Glutamax and 20 mM HEPES, 1 mM Na-pyruvate, non-essential amino acids 1X (all from Life Technologies), 100 μg/mL streptomycin and 100 μ/mL penicillin (GE Healthcare) and 37.5 μM β-mercaptoethanol (Sigma-Aldrich) supplemented with 20% heat-inactivated fetal calf serum (FCS) (Life Technologies)). For phorbol-myristate-acetate (PMA) and ionomycin stimulation experiments, whole splenocytes were stimulated or not with 40 nM of PMA and 800 nM ionomycin for 4 h in the presence of 2 µg/mL of brefeldin A (all from Sigma Aldrich, Merck) and cultured at 37 °C and 5% CO2. For IL-3, IL-4 or anti-IgE stimulations, cells were stimulated with the doses indicated in the figure legends for 2 or 20 h at 37 °C and 5% CO2. Then, cells were harvested by repeated flushing, and wells were washed with 1 mL of PBS. Samples were then prepared for flow cytometry analysis.
5
Differentiating hiPSC-derived OPCs into Oligodendrocytes
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To differentiate human induced pluripotent stem cells derived OPCs (Tempo Biosciences) into mature, myelin-positive oligodendrocytes (MBP) we plated OPCs on matrigel (Corning) and incubated them with DMEM/F12 containing HEPES, glutamine (2 mM), nonessential amino acids (1X) (Life Tech), 1X StemPro Neural Supplement (Invitrogen), PDGF-AA (10 ng/mL, Cat: 100-13A, Peprotech), NT3, (10 ng/mL, Cat: 450-03, Peprotech), biotin (100 ng/mL, Sigma-Aldrich), and cAMP (5 µM, Sigma-Aldrich). Cell differentiation was induced for 30 days following incubation with 50:50 DMEM/F12:Neurobasal medium (Life Tech) containing nonessential amino acids 1X (Life Tech), 1X B27 (Life Tech), 2 l -glutamine (Life Tech), biotin (100 ng/mL, Sigma-Aldrich), brain-derived neurotrophic factor (BDNF, 10 ng/mL; Cat: 450-02, Peprotech), ascorbic acid (20 μg/mL, Sigma-Aldrich), cAMP (1 μM, Sigma-Aldrich), and T3 (200 ng/mL, Sigma-Aldrich).
6
Mouse Embryonic Explant Culture
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Explant culture was performed as previously described4 (link). LuVeLu CD1 E9.5 mice (both male and female) were sacrificed according to local regulations in agreement with national and international guidelines. We complied with all relevant ethical regulations. Study protocol was approved by Brigham and Women’s Hospital IACUC/CCM (Protocol number N000478). Sample size was not estimated, nor were randomization or blinding performed. Tailbud was dissected with a tungsten needle and ectoderm was removed using Accutase (Life Technologies). Explants were then cultured on fibronectin-coated plate (LabTek chamber). The medium consists of DMEM, 4.5g/L Glucose, 2mM L-Glutamine, non-essential amino acids 1x (Life Technologies), Penicillin 100U/mL, Streptomycin 100μg/mL, 15% fetal bovine serum (FBS), Chir-99021 3μM, LDN193189 200nM, BMS-493 2.5 μM, mFgf4 50ng/mL, heparin 1μg/mL, HEPES 10mM and Y-27632 10μM. Explants were incubated at 37°C, 7.5% CO2. Live imaging was performed on a confocal microscope Zeiss LSM 780, using a 20X objective (note that the tiling could create lines between the different images). For micropattern culture, explants were cultured overnight in standard condition, then dissociated using Trypsin-EDTA, and plated on fibronectin-coated CYTOOchips Arena in a CYTOOchamber 4 wells.
7
Culturing Mouse Embryonic Stem Cells and Fibroblasts
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Bruce 4 (C57/B6 Strain) mouse embryonic stem cells (ESCs) and HEK293T were purchased from ATCC. Mouse Embryonic Fibroblasts (MEFs) were isolated as previously described [8 (link)]. Briefly, E13.5 C57BL/6 mouse embryos from 13.5 day-pregnant female mice were placed in (1X) PBS, the head and embryonic internal organs were dissociated from the abdominal cavity, then trypsinized and passed through a 10-ml syringe to produce single-cell suspensions, which were further expanded. MEFs and HEK293T cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) Sigma-Aldrich, Cat. No. D6429) supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS), Penicillin/Streptomycin (100 ug/ml; Thermo Fischer Scientific, Cat. No. 15140122), 2mM GlutaMAX (Gibco #,Cat. No. 35-050-061), and 0.1 mM Non-Essential Amino Acids 100X (Gibco, Cat. No. 11140050), at 37°C and 5% CO2. Bruce4 ESCs were cultured in complete ESC medium [Knock Out MediumDMEM (Gibco, Cat. No. 10829018), 20% ES-tested FBS (Pansera, Cat. No. P30-2602), Penicillin/Streptomycin 1X (Thermo Fischer Scientific, Cat. No. 15140122), GlutaMAX 1X (Gibco, Cat. No. 35-050-061), and Non-Essential Amino Acids 1X (Gibco, Cat. No. 11140050)], with 20 ng/mL mLIF (Santa Cruz Biotechnology, sc-4378).
8
Dermal Fibroblast Cultures from Abcc6 Mice
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Dermal fibroblast cultures were obtained from the whole skin of 37 Abcc6−/− and 29 Abcc6+/+ mice of 0.5 (n=27) and 12 months (n=39) of age. The study was approved by the Ethical Committee of the University of Modena and Reggio Emilia. Fibroblasts were cultured in DMEM (Gibco, Grand Island, New York, USA) with 10% fetal bovine serum (FBS) (Lonza) and used at 2nd or 3rd passages. During all experimental procedures, fibroblasts were pooled from several animals of the same age and genotype. Cells were routinely cultured in 75cm2 flasks (Nunc, Roskilde, Denmark) with DMEM supplemented with 10% FBS, penicillin 100 IU/ml, streptomycin 100 μg/ml and nonessential amino acids 1X (Gibco). Cells were observed by phase contrast microscopy.
9
Dermal Fibroblast Cultures from Abcc6 Mice
10
Characterization of Human Neural Stem Cells
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Cell line hNS1 was used as a model of human neural stem cells (hNSCs) that has been previously characterized [35 (link)–38 (link)]. This cell line is non-transformed, derived from human fetal forebrain and immortalized with v-myc [39 (link)]. Cells were cultured on poly-L-lysine (10 μg/mL; Sigma) coated plastic plates and proliferated in human stem cell (HSC) medium [Dulbecco’s Modified Eagle Medium (DMEM)/F12 with GlutaMAX-I medium (Gibco) containing 0.26% AlbumaMAXb (Gibco), 0.6% glucose (Merck), N2 Supplement 1X (Gibco), 5 mM HEPES (Gibco), penicillin/ streptomycin 1× (P/S; Lonza), non-essential aminoacids 1X (Gibco)] and supplemented with 20 ng/mL epidermal growth factor (EGF; PreproTech) and 20 ng/mL basic fibroblast growth factor (FGF2; PreproTech). hNS1 cells were differentiated by withdrawal of growth factors (EGF and FGF2) and addition of 0.5% heat-inactivated fetal bovine serum (FBS) (differentiation medium). Cells were kept in an incubator set to 37 °C and 5% CO2.
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