Non essential amino acid (neaa)

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Non-essential amino acids are a group of amino acids that can be synthesized by the human body, and are not required to be obtained from dietary sources. They play a fundamental role in protein synthesis and other metabolic processes.

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Non-essential amino acids (4961 citations) Essential amino acids (37 citations) Amino acids (13 citations) MEM Non-Essential Amino Acids Solution (NEAA, (11 citations) Non-essential amino acids (NEAA; (11 citations) Non-Essential Amino Acids Solution (NEAA) (6 citations) MEM Non-Essential Amino Acids (NEAAs; (4 citations) Eagle’s minimum essential medium non-essential amino acids (MEM NEAA) (2 citations) MEM Non-Essential Amino Acids Solution (MEM-NEAA, (2 citations)

154 protocols using non essential amino acid (neaa)

Fibroblast-derived iPSC Hematopoietic Differentiation

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Human dermal adult forearm fibroblasts were cultured in Fib media (Dulbecco’s modified Eagle’s medium [DMEM] with 10% vol/vol fetal bovine serum [Neonatal Bovine Serum, HyClone, Logan, UT, http://www.hyclone.com], 1% vol/vol nonessential amino acid [Gibco, Grand Island, NY, http://www.invitrogen.com], and 1 mM l-glutamine [Gibco]). Patient-specific iPSCs were derived and cultured on irradiated mouse embryonic fibroblasts (iMEFs) in F-12 iPSC media (DMEM/F-12 [Gibco] with 20% knockout serum replacement [Gibco], 100 µM β-mercaptoethanol, 100 µM nonessential amino acid [Gibco], 1 mM l-glutamine [Gibco]) supplemented with 10 ng/ml basic fibroblast growth factor; F-12 iPSC media were not supplemented with antibiotics. iPSC-derived embryoid bodies (EBs) were cultured in hematopoietic differentiation media (KO-DMEM [Gibco] with 20% knockout serum replacement [Gibco], 100 µM β-mercaptoethanol, 100 µM nonessential amino acid [Gibco], 1 mM l-glutamine [Gibco]) supplemented with 50 ng/ml granulocyte colony-stimulating factor (Amgen, Inc., Thousand Oaks, CA, http://www.amgen.com), 300 ng/ml stem cell factor (Amgen, Inc.), 10 ng/ml interleukin-3 (IL-3; R&D Systems, Minneapolis, MN, http://www.rndsystems.com), 10 ng/ml interleukin-6 (IL-6; R&D Systems), 25 ng/ml bone morphogenetic protein 4 (BMP4; R&D Systems), and 300 ng/ml Flt-3 ligand (Flt-3L; R&D Systems).

Salci K.R., Lee J.H., Laronde S., Dingwall S., Kushwah R., Fiebig-Comyn A., Leber B., Foley R., Dal Cin A, & Bhatia M. (2015). Cellular Reprogramming Allows Generation of Autologous Hematopoietic Progenitors From AML Patients That Are Devoid of Patient-Specific Genomic Aberrations. Stem Cells (Dayton, Ohio), 33(6), 1839-1849.

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Cell Culture Conditions for Cancer Cell Lines

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HepG2, FOCUS, Hep3B, Mahlavu, Huh7, Hep40,
and PLC-PRF-5 hepatocellular carcinoma cells; MCF-7, MDA-MB-231, MDA-MB-468,
SKBR3, and ZR-75 breast cancer cells; and normal-like epithelial MCF-10A
breast cells were cultured in low-glucose Dulbecco’s modified
Eagle’s medium (DMEM) (Biological Industries-BI) supplemented
with 10% fetal bovine serum (FBS) (Gibco/Thermo Fisher Scientific),
2 mM l-Glutamine (Gibco/Thermo Fisher Scientific), 100 Units/mL
Penicillin/Streptomycin, and 0.1 mM non-essential
amino acid (Gibco/Thermo Fisher Scientific). SNU475 hepatocellular
carcinoma cells were maintained in RPMI (Biological Industries-BI)
supplemented with 10% fetal bovine serum (FBS) (Gibco/Thermo Fisher
Scientific), l-Glutamine (Gibco/Thermo Fisher Scientific),
and 100 Units/mL Penicillin/Streptomycin, while ZR-75 breast cancer
cells were cultured RPMI (Biological Industries-BI) supplemented with
10% fetal bovine serum (FBS) (Gibco/Thermo Fisher Scientific), 1×
sodium pyruvate, and %4.5 glucose. Normal-like MCF-10A breast cells
were sustained in DMEM/HAM’S F12 (Hyclone) supplemented with
10% fetal bovine serum (FBS) (Gibco/Thermo Fisher Scientific), EGF
(20 ng/mL), Hydrocortisone (0.5 mg/mL), cholera toxin (100 ng/mL),
insulin (10 μg/mL), and 100 Units/mL Penicillin/Streptomycin.
The cells were cultivated at 37 °C in a humidified incubator
under 5% CO₂.

Turanlı S., Nalbat E., Lengerli D., İbiş K., Güntekin Ergün S., Akhan Güzelcan E., Muyan M., Cetin-Atalay R., Çalışkan B, & Banoglu E. (2022). Vicinal Diaryl-Substituted Isoxazole and Pyrazole Derivatives with In Vitro Growth Inhibitory and In Vivo Antitumor Activity. ACS Omega, 7(41), 36206-36226.

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4sU Labeling of Mouse Embryonic Stem Cells

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Wild-type (WT) and Tet-TKO J1 mESCs (ATCC, SCRC-1010) were cultured in presence of Mitomycin C inactivated mouse embryonic fibroblasts on 0.1% gelatin-coated (Millipore, ES-006-B) 6-well plates in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, 11965084) supplemented with 15% fetal bovine serum (Gibco, 16000044), 0.1 mM nonessential amino acid (Gibco, 11140050), 1 mM sodium pyruvate (Gibco, 11360070), 2 mM L-glutamine (Gibco, 25030081), 50 μM 2-mercaptoethanol (Gibco, 31350010), 1 μM MEK inhibitor PD0325901 (Axon Med Chem, Axon 2128) and 3 μM GSK3 inhibitor CHIR99021 (Axon Med Chem, Axon 2128), and 1,000 U/mL LIF (Gemini Bio-Products, 400–495-7). Cells were maintained at 37°C with 5% CO₂ and passage every 2–3 days. The average doubling time of J1 mESCs in presence of 4sU as determined by cell counting was 14.8 hours.
For labeling experiments, 4sU (Alfa Aesar, J60679) were dissolved in DMSO to make 1 M stock. WT and Tet-TKO mESCs were seeded at a density of 3×10⁵ cells/mL two days before the labeling experiments and cultured in feeder-free conditions (0.1% gelatin-coated plates). One timepoint 4sU labeling was performed by incubating mESCs in fresh medium supplemented with 4sU (at a final concentration of 100 μM). After 4 hours of labeling, mESCs were rinsed once with PBS and dissociated into single cell suspensions with TrypLE-Express (Gibco, 12605010) for 5 min at 37°C.

Qiu Q., Hu P., Qiu X., Govek K.W., Camara P.G, & Wu H. (2020). Massively parallel and time-resolved RNA sequencing in single cells with scNT-Seq. Nature methods, 17(10), 991-1001.

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Culturing Human Osteosarcoma Cell Lines

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Human osteosarcoma cell lines MG63 and U2OS were purchased from Cell Resource Center of Shanghai Institutes for Biological Sciences (Shanghai, China) and cultured in Minimal Essential Medium (Gibco, Waltham, Massachusetts, USA) with 10% fetal bovine serum (Biological Industries, Kibbutz Beit Haemek, Israel), 1% non-essential amino acid (Gibco), and penicillin/streptomycin (Gibco) in a humidified incubator under 95% air and 5% CO₂ at 37 °C. All other cell culture materials were obtained from Gibco; all chemicals were obtained from Sigma-Aldrich (St. Louis, Missouri, USA).

Wang D.Y., Wu Y.N., Huang J.Q., Wang W., Xu M., Jia J.P., Han G., Mao B.B, & Bi W.Z. (2016). Hippo/YAP signaling pathway is involved in osteosarcoma chemoresistance. Chinese Journal of Cancer, 35, 47.

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Cell Culture Conditions for Various Cell Lines

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TPC-1 cells were cultured in DMEM supplemented with 5% FBS and 2 mmol/l l-glutamine (all from Gibco, Carlsbad, CA). WRO cells were maintained in RPMI 1640 medium (Gibco) supplemented with 10% FBS, 2 mmol/l l-glutamine, 1 mmol/l sodium pyruvate (Gibco), and 1 mM nonessential amino acid (Gibco). COS-7 cells and human bronchial epithelial BEAS2B cells (ATCC, Manassas, VA) were cultured in low-glucose DMEM (Life Technologies, Rockville, MD) with 10% FBS (Gibco). All cells were cultured at 37°C with 5% CO2 in a humidified cell incubator.

Moodley S., Hui Bai X., Kapus A., Yang B, & Liu M. (2015). XB130/Tks5 scaffold protein interaction regulates Src-mediated cell proliferation and survival. Molecular Biology of the Cell, 26(24), 4492-4502.

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Murine Macrophage Stimulation and Modulation

Cited 1 time

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We used the murine macrophage cell line Raw 264.7 in this study. Cells were cultured in RPMI 1640 (Biowest, MO, USA) containing 10% fetal bovine serum (Biowest), 10% non-essential amino acid (Gibco, Carlsbad, CA, USA), 1% 2-mercaptoethanol (Gibco), and 10% penicillin (Gibco). Cells were maintained at 37°C in humidified air with 5% carbon dioxide. They were washed twice with pH 7.4 phosphate-buffered saline (PBS; Gibco) before treatment. Cells were incubated with 0.1 μg lipopolysaccharide (LPS) for 4 h, followed by incubation with 2 mM ATP for 2 h before treatment with 10 μM rosuvastatin (Sigma Aldrich, St. Louis, MO, USA) and/or 10 μM olmesartan (Sigma Aldrich). The dose of each drug was based on the results of previous studies [15 (link)–18 (link)].

Lee S.G., Lee S.J., Thuy N.V., Kim J.S., Lee J.J., Lee O.H., Kim C.K., Oh J., Park S., Lee O.H., Kim S.H., Park S., Lee S.H., Hong S.J., Ahn C.M., Kim B.K., Ko Y.G., Choi D., Hong M.K, & Jang Y. (2019). Synergistic protective effects of a statin and an angiotensin receptor blocker for initiation and progression of atherosclerosis. PLoS ONE, 14(5), e0215604.

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Melanoma Cell Line and T Cell Isolation

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Melanoma cell lines were generated from patients with histologically proven melanoma. HLA-A2pos/TAP-deficient T2 cells were obtained from the ATCC (CRL-1992). Peripheral blood mononuclear cells (PBMC) were isolated from fresh whole blood using LymphoprepTM (Axis-Shield Density Gradient Media). Culture medium (HS medium) was prepared as follows: RPMI supplemented with penicillin/streptomycin (Gibco), L-glutamine (Gibco), non-essential amino acid (Gibco), Na pyruvate (Gibco), Kanamycin (Gibco), 2β-mercaptoethanol (Sigma) and 8% of human serum (HS). In some experiments, CD4 + T cells and CD8 + T cells were positively isolated with magnetic beads (Miltenyi Biotec).

Gestermann N., Saugy D., Martignier C., Tillé L., Fuertes Marraco S.A., Zettl M., Tirapu I., Speiser D.E, & Verdeil G. (2020). LAG-3 and PD-1+LAG-3 inhibition promote anti-tumor immune responses in human autologous melanoma/T cell co-cultures. Oncoimmunology, 9(1), 1736792.

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Dengue Virus 2 Culture Protocol

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Dengue serotype 2 New Guinea C strain virus (DENV-2 NGC) was obtained from the Whitehead lab at the National Institutes of Health, passaged in monolayers of C6/36 cells, and a virus stock at a concentration of 7.48 log₁₀ plaque-forming units (pfu)/mL was frozen in multiple aliquots at –80°C. Ae. albopictus C6/36 cells were cultured in Minimum Essential Medium (MEM) supplemented with 10% FBS, 2 mM L-glutamine (Gibco), 2 mM non-essential amino acid (Gibco) and 0.05 mg/mL gentamycin (Invitrogen) and were maintained at 32°C, 80% RH in 5% CO₂.

Tsujimoto H., Hanley K.A., Sundararajan A., Devitt N.P., Schilkey F.D, & Hansen I.A. (2017). Dengue virus serotype 2 infection alters midgut and carcass gene expression in the Asian tiger mosquito, Aedes albopictus. PLoS ONE, 12(2), e0171345.

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Somatic Cell Nuclear Transfer in Mice

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Female MEFs generated from FVB/B6 (FVB/N females x C57BL/6 males), B6/FVB (C57BL/6 females x FVB/N males), or homozygous FVB/N mice and adult fibroblasts isolated from homozygous FVB/N female mice were used as the donor cells for SCNT. MEFs were established from embryos at 13.5 dpc and the embryos’ heads and organs were removed before cell isolation. Adult fibroblasts were isolated from ear skin tissue at 8 weeks old. Tissue was dissociated with 0.1% collagenase IV, incubated for 30 min, and diluted with an equal volume of F12/DMEM media (Gibco10099141) supplemented with 10% FBS (Gibco). The cells were cultured in F12/DMEM with 10% FBS, 100 units/ml penicillin (Hyclone), 100 μg/ml streptomycin (Hyclone), 100 μM β-mercaptoethanol (Sigma), and 100 μM nonessential amino acid (Gibco) under 5% CO₂ at 37 °C in a humidified incubator. Cumulus cells were collected from BDF1 or FVB/N mice and kept in KSOM (Merck) until nuclear transfer.

Lee Y., Trout A., Marti-Gutierrez N., Kang S., Xie P., Mikhalchenko A., Kim B., Choi J., So S., Han J., Xu J., Koski A., Ma H., Yoon J.D., Van Dyken C., Darby H., Liang D., Li Y., Tippner-Hedges R., Xu F., Amato P., Palermo G.D., Mitalipov S, & Kang E. (2022). Haploidy in somatic cells is induced by mature oocytes in mice. Communications Biology, 5, 95.

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Constructing NTD Mutant Cell Lines for Deep Mutational Scanning

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HEK293T landing pad cells (37 (link), 38 (link)) were used to display the NTD mutant library for deep mutational scanning. Landing pad cells were maintained using complete growth medium consisting of Dulbecco’s modified Eagle’s medium (DMEM; Corning), 10% (v/v) fetal bovine serum (FBS; VWR), penicillin-streptomycin (Gibco), nonessential amino acid (Gibco), and doxycycline (2 μg/ml). Plasmid (1.2 μg) was transfected into 6 × 10⁵ landing pad cells. For the deep mutational scanning experiment, eight transfection reactions were carried out in parallel to minimize loss of mutant diversity at the transfection step. Transfected cells were then incubated at 37°C with 5% CO₂. After 48 hours, 10 nM AP1903 was supplemented to carry out negative selection. At 72 hours after the negative selection, positive selection antibiotic [puromycin (1 μg/ml) for NTD cell lines or hygromycin (100 μg/ml) for hACE2 cell lines] was supplemented to the medium to carry out positive enrichment of cells with successful recombination. Constructed cell lines would remain in the complete growth medium supplemented with doxycycline and the positive selection antibiotics.

Ouyang W.O., Tan T.J., Lei R., Song G., Kieffer C., Andrabi R., Matreyek K.A, & Wu N.C. (2022). Probing the biophysical constraints of SARS-CoV-2 spike N-terminal domain using deep mutational scanning. Science Advances, 8(47), eadd7221.

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