We used a hiPSC line; 201B6[4 (link)] for generating cardiovascular cell populations. The methods for culturing and passaging human iPSCs have been previously reported in detail [8 (link)]. Briefly, iPSCs were detached with Versene (0.48 mM EDTA solution; Life Technologies, Carlsbad, CA, USA) and plated onto Matrigel (growth factor reduced, 1:60 dilution; Life Technologies)-coated plates at a density of approximately 100,000 cells/cm2 in mouse embryonic fibroblast conditioned medium [MEF-CM; Dulbecco’s modified Eagle’s medium (DMEM) (Nacalai Tesque, Kyoto, Japan) containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 1% non-essential amino acids (NEAA) (Life Technologies)] with 4 ng/ml bFGF (Wako Pure Chemicals Industries, Osaka, Japan) for 2 days before induction. Cells were covered with Matrigel (1:60 dilution) 1 day before induction. MEF-CM was replaced with RPMI+B27 medium (RPMI1640, 2 mM L-glutamine, B27 supplement without insulin; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 100 ng/ml of Activin A (R&D, Minneapolis, MN, USA) for 1 day, followed by 10 ng/ml BMP4 (R&D) and 10 ng/ml bFGF for 3 days without culture medium change. At 5 days of differentiation, the culture medium was replaced with RPMI+B27 medium with 50 ng/ml VEGF165 (Wako Pure Chemicals Industries), and culture medium was refreshed every other day.
Non essential amino acids neaa
Manufactured by Thermo Fisher Scientific
Sourced in Japan, Germany
Non-essential amino acids (NEAA) are a group of amino acids that can be synthesized by the human body and are not required to be obtained through dietary sources. These amino acids play a crucial role in various physiological processes within the body.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
Dmem f12, by Thermo Fisher Scientific (4 mentions)
β mercaptoethanol, by Merck Group (4 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (3 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Basic fibroblast growth factor (bfgf), by Fujifilm (1 mentions)
Versene, by Thermo Fisher Scientific (1 mentions)
Vegf165, by Fujifilm (1 mentions)
Matrigel, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium dmem, by Nacalai Tesque (1 mentions)
Basic fibroblast growth factor (bfgf), by R&D Systems (1 mentions)
Activin a, by R&D Systems (1 mentions)
Bone morphogenetic protein 4 (bmp4), by R&D Systems (1 mentions)
Shark chondroitin sulfate, by Merck Group (1 mentions)
Resistin, by BioVendor (1 mentions)
Recombinant human adiponectin, by BioVendor (1 mentions)
Calf thymus dna, by Merck Group (1 mentions)
Proline, by Merck Group (1 mentions)
11 protocols using non essential amino acids neaa
1
Cardiac Differentiation of Human iPSCs
2
Bovine Chondrocyte Isolation and Culture
3
Culturing Human Embryonic Kidney and Pluripotent Stem Cells
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Human embryonic kidney 293T (HEK293T) cells were cultured in Dulbecco’s Eagle Medium (DMEM) (Life Technologies), 10% fetal bovine serum (FBS) (Gibco), and 1× Penicillin/Streptomycin (Life Technologies). HEK293T cells were from ATCC (CRL-3216).
Undifferentiated hPSCs were cultured in hPSC medium: DMEM/F12 (Life Technologies), 20% KnockOut Serum Replacement (KSR) (Life Technologies), 1× Non-Essential Amino Acids (NEAA) (Life Technologies), 0.055 mM 2-mercaptoethanol (Sigma), 1× Penicillin/Streptomycin, and 10 ng ml−1 bFGF (Peprotech). hPSCs were maintained on CF1 feeder cells at 37 °C and 5% CO2. hPSCs were isolated by Accutase (Life Technologies) as 1:3 to 1:6 every 3–6 days. 0.5 μM thiazovivin (TargetMol) were used during the first 24 h when passaging or thawing cells. MEL1 INSGFP/W hESC line46 (link) was a kind gift from Drs. E. G. Stanley and Andrew Elefanty. Mycoplasma contamination was routinely detected using TransDetect PCR Mycoplasma Detection Kit (TransGen Biotech).
Undifferentiated hPSCs were cultured in hPSC medium: DMEM/F12 (Life Technologies), 20% KnockOut Serum Replacement (KSR) (Life Technologies), 1× Non-Essential Amino Acids (NEAA) (Life Technologies), 0.055 mM 2-mercaptoethanol (Sigma), 1× Penicillin/Streptomycin, and 10 ng ml−1 bFGF (Peprotech). hPSCs were maintained on CF1 feeder cells at 37 °C and 5% CO2. hPSCs were isolated by Accutase (Life Technologies) as 1:3 to 1:6 every 3–6 days. 0.5 μM thiazovivin (TargetMol) were used during the first 24 h when passaging or thawing cells. MEL1 INSGFP/W hESC line46 (link) was a kind gift from Drs. E. G. Stanley and Andrew Elefanty. Mycoplasma contamination was routinely detected using TransDetect PCR Mycoplasma Detection Kit (TransGen Biotech).
4
Maintenance of Pluripotent H9 Cells
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H9 cells were cultured on a layer of mouse embryonic fibroblast cells (MEFs) in hESC medium consisting of DMEM/F12 (Life Technologies, Carlsbad, MA) with 20% KnockOut Serum Replacer (Life Technologies), 0.1 mM Non-essential Amino Acids (NEAA, Life Technologies), 1 mM L-glutamine (Life Technologies), and 0.1 mM β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO) and then supplemented with 4 ng/ml bFGF (Life Technologies).
5
Isolation and Expansion of Bone Marrow-Derived Mesenchymal Stem Cells
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BMSCs were isolated from bone marrow washouts as described previously [22 (link),23 (link),24 (link)]. In short, bone marrow was collected in a heparinized syringe and cells were fractioned on a Ficoll-Paque Plus density gradient (GE Healthcare Europe, Freiburg, Germany). The fraction of mononuclear cells containing the BMSCs was washed in PBS (Life Technologies, Darmstadt, Germany) and seeded in several 0.1% gelatin-coated (Sigma-Aldrich, Steinheim, Germany) T75 cell culture flasks (Nunc, Roskilde, Denmark).
Cells were cultivated in expansion medium composed of DMEM high glucose supplemented with 12.5% FCS, 1% penicillin/streptomycin, 1% L-glutamine (Life Technologies, Darmstadt, Germany), 1% non-essential amino acids (NEAA; Life Technologies, Darmstadt, Germany), 0.1% β-mercaptoethanol (Life Technologies, Darmstadt, Germany) and 4 ng/mL fibroblast growth factor 2 (Abcam, Cambridge, U.K.). To discard non-adherent cells, medium was changed after the first 24 h and subsequently twice weekly. Cells were passaged at 80% confluency and stored in liquid nitrogen until further use in passage 4.
Cells were cultivated in expansion medium composed of DMEM high glucose supplemented with 12.5% FCS, 1% penicillin/streptomycin, 1% L-glutamine (Life Technologies, Darmstadt, Germany), 1% non-essential amino acids (NEAA; Life Technologies, Darmstadt, Germany), 0.1% β-mercaptoethanol (Life Technologies, Darmstadt, Germany) and 4 ng/mL fibroblast growth factor 2 (Abcam, Cambridge, U.K.). To discard non-adherent cells, medium was changed after the first 24 h and subsequently twice weekly. Cells were passaged at 80% confluency and stored in liquid nitrogen until further use in passage 4.
6
Murine and Human iPSC Generation
7
Feeder-Dependent Culture of Spermatogonial Stem Cells
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The srFGSCs and faFGSCs culture system uses a mitotically inactivated STO feeder layer. STO cells were cultured in Dulbecco’s modified Eagle’s medium with high-glucose (Life Technologies), 1 mM non-essential amino acids (NEAA; Life Technologies), 10% FBS (Hyclone), and 6 mg/L penicillin (Sigma). The STO cells were treated with 10 μg/mL mitomycin C (Sigma) for 2–3 h, then washed several times in HBSS, digested to single cells, and plated on 0.1% (w/v) gelatin-coated wells of a 24-well plate. The culture medium for srFGSCs and faFGSCs consist of minimum essential medium α (MEMα), 10% FBS (Front), 1 mM sodium pyruvate (Sigma), 1 mM NEAA, 2 mM L-glutamine (Sigma), 0.1 mM β-mercaptoethanol (Sigma), 10 ng/mL human leukemia inhibitory factor (LIF; Santa Cruz Biotechnology, sc-4377), 10 ng/mL human epidermal growth factor (EGF; Peprotech, AF-100-15), 40 ng/mL human glial cell line-derived neurotrophic factor (GDNF; Peprotech, 450-10), 10 ng/mL human basic fibroblast growth factor (bFGF; Peprotech, AF-100-18B), and 6 mg/L penicillin. FGSCs were cultured on STO feeders in FGSC culture medium (500 μL per well). The medium was changed every 2–3 days and cells were subcultured using dispase II (Roche) every 5–8 days at a 1:1–1:3 dilution. All cultures were maintained at 37 °C in a 5% CO2-95% air atmosphere.
8
Human Metapneumovirus Cell Culture Protocol
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LLC-MK2 cells (ATCC CCL-7) were maintained in minimal essential medium (MEM) (Life Technologies) supplemented with 10% fetal bovine serum (FBS) (Wisent).
BSR-T7/5 cells (a gift from Dr Ursula Buchholz at the NIAID in Bethesda, MD) were cultured in MEM supplemented with 10% FBS (Wisent), 1% Non-essential amino acids (NEAA) (Life Technologies), 10 mM HEPES (sigma), 1% penicillin / streptomycin (Wisent) and 0.2 mg/ml geneticin (G418, Life Technologies). The HMPV group A strain C-85473 and group B strain CAN98–75 were grown on LLC-MK2 cells in OptiMEM (Life technologies) supplemented with 0.0002% trypsin (Sigma). Virus stocks were concentrated on Amicon columns (Fisher Scientific) as previously described [20 (link)].
BSR-T7/5 cells (a gift from Dr Ursula Buchholz at the NIAID in Bethesda, MD) were cultured in MEM supplemented with 10% FBS (Wisent), 1% Non-essential amino acids (NEAA) (Life Technologies), 10 mM HEPES (sigma), 1% penicillin / streptomycin (Wisent) and 0.2 mg/ml geneticin (G418, Life Technologies). The HMPV group A strain C-85473 and group B strain CAN98–75 were grown on LLC-MK2 cells in OptiMEM (Life technologies) supplemented with 0.0002% trypsin (Sigma). Virus stocks were concentrated on Amicon columns (Fisher Scientific) as previously described [20 (link)].
9
Generation of hESCs with Intronic Mutation
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hESCs (H9, line WA09 (WiCell), passages 20–40) were cultured on a feeder layer of irradiated mouse embryonic fibroblasts in a humidified incubator with 5% CO2 at 37 °C. The hESC medium containing DMEM/F12 (Gibco), 20% Knock Serum Replacement (Gibco, 10828010), 0.1 mmol/L beta-mercaptoethanol (Sigma), 1% (v/v) Non-Essential Amino Acids (NEAA, Life Technologies), and 0.5% (v/v) GlutaMAX (Life Technologies). The medium was changed every day with 10 ng/mL of basic fibroblast growth factor (PeproTech). hESCs were passaged every 7 days. hESCs with an intronic mutation (8-61757392-C-T) were transformed from H9 hESCs by introducing an intronic mutation and two synonymous mutations by homology-mediated end joining-based targeted integration using CRISPR/Cas9. The editor vector (Addgene #48138) containing the small guiding RNA (sgRNA) that targeting exon sequence of CHD7, together with the donor vector containing a 1.6 kB homologous sequence of CHD7 and carrying an intronic mutation and two synonymous mutations, were transfected into H9 hESCs by liposome. Single GFP+ cells were isolated using flow cytometry and cultured. The cells with an intronic point mutation were identified by DNA sequencing.
10
Adoptive Transfer of OT-I CD8+ T Cells
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CD45.1+/− OT-I mice were a gift of Y. Kim at Vanderbilt University Medical Center. 6–8 week C57BL/6 CD45.1+/− OT-I mice were euthanized and spleens were harvested using EasySep Mouse CD8+ T Cell Isolation Kit (STEMCELL Technologies). Briefly, T cells were activated in vitro in supplemented RPMI 1640 (Gibco) with 10% HI-FBS (Gibco), 1% penicillin/streptomycin (Gibco), 50 μM ß-mercaptoethanol (MilliporeSigma), 1 mM sodium pyruvate, minimum essential medium NEAA (non-essential amino acids) (Gibco), 10 mM HEPES (Gibco), recombinant mouse interleukin-2 (10 U/ml; MilliporeSigma), and Dynabeads Mouse T-Activator CD3/CD28 (at a bead-to-cell ratio of 1:1; Gibco) at 37 °C in a CO2 incubator (5%). After 5 days, T cells were magnetically separated from Dynabeads and allowed to rest for 24 h before use. The following day, in the B16.F10-OVA model, 5 × 105 OT-I CD8+ T cells were adoptively transferred by retro-orbital injection.
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