Mem neaa

Manufactured by Thermo Fisher Scientific

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MEM NEAA is a cell culture medium supplement that provides a source of non-essential amino acids for cell growth and maintenance. It is designed to be added to Minimum Essential Medium (MEM) to support the nutritional requirements of various cell types.

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MEM NEAA (100×), (12 citations) MEM NEAA 100x (12 citations) MEM Non-Essential Amino Acids Solution (NEAA, (11 citations) MEM nonessential amino acids (NEAA; (10 citations) MEM Non-Essential Amino Acids (NEAAs; (4 citations) MEM NEAA supplement (4 citations) MEM nonessential amino acids (MEM NEAA) (3 citations) MEM-Non-Essential Amino Acid solution (NEAA) (2 citations) MEM Non-Essential Amino Acids Solution (MEM-NEAA, (2 citations) MEM nonessential amino acids (NEAA) solution (2 citations)

399 protocols using mem neaa

Neural Cell Culture Media Formulations

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MEF media: DMEM, 10% FBS, 100 U/ml Penicillin-Streptomycin, 2 mM l-glutamine (Life Technologies).

iPSC media: DMEM/F12, 20% KOSR, 1× MEM-NEAA, 1× Penicillin-Streptomycin-Glutamine, 55 µM beta- mercaptoethanol (Life Technologies).

N2 neural induction media: DMEM/F12, 1× N2 supplement, 1× MEM-NEAA (Life Technologies), 2 µg/ml heparin (Sigma).

N2/B27 neural induction media: DMEM/F12, 1× N2 supplement, 1× B27 supplement, 1× MEM-NEAA (Life Technologies), 2 µg/ml heparin (Sigma).

Neural differentiation media: Neurobasal medium, 1× N2 supplement, 1× B27 supplement, 1× MEM-NEAA (Life Technologies), 2 µg/ml heparin (Sigma).

Srikanth P., Lagomarsino V.N., Pearse RV I.I., Liao M., Ghosh S., Nehme R., Seyfried N., Eggan K, & Young-Pearse T.L. (2018). Convergence of independent DISC1 mutations on impaired neurite growth via decreased UNC5D expression. Translational Psychiatry, 8, 245.

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Porcine MSCs Adipogenic Differentiation

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Porcine mesenchymal stem cells were isolated from adipose tissue and bone marrow from a 3-month-old Polish Large White pig, as described by Kociucka et al. (2016 (link)). The cells were cultured in advanced DMEM medium (Gibco) supplemented with 10% FBS (Sigma-Aldrich), 5 ng/ml FGF-2 (PromoKine), 2 mM L-Glutamine (PAA), 1 mM 2-mercaptoethanol (Sigma-Aldrich), 1 × antibiotic antimycotic solution (Sigma Aldrich), and MEM NEAA (Thermo Fisher Scientific) at 37 °C with 5% CO₂ supplementation. To induce adipogenic differentiation, the cells were grown to confluency and were cultured with adipogenic differentiation medium composed of advanced DMEM (Gibco) with 10% (v/v) FBS (Sigma), 1 × antibiotic antimycotic solution (Sigma Aldrich), MEM NEAA (Thermo Fisher), 5 ng FGF-2 (PromoKine), 1 × linoleic acid albumin (Sigma-Aldrich), 1 × ITS (Sigma-Aldrich), 1 μM dexamethasone (Sigma-Aldrich), 100 μM indomethacin (Sigma-Aldrich), and 50 mM IBMX (Sigma-Aldrich). The differentiation process was allowed to proceed for 7 days and lipid droplet formation was examined using a phase-contrast microscope (TS100 Eclipse, Nikon).

Stachecka J., Nowacka-Woszuk J., Kolodziejski P.A, & Szczerbal I. (2019). The importance of the nuclear positioning of the PPARG gene for its expression during porcine in vitro adipogenesis. Chromosome Research, 27(3), 271-284.

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BRAF V600E and PIK3CA H1047R Mutant ATC Cell Lines

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The BRAF^V600E mutant ATC cell lines 8505c and SW1736 were purchased at the Public Health England repository and cultured in RPMI-1640, 10% FCS, 2 mM L-glutamine, MEM NEAA (Thermo Fisher) 1:100 and P/S (100U Penicilin/ml and 0.1mg Streptomycin/ml). The ATC cell line OCUT-2 carries concomitant BRAF^V600E and PIK3CA^H1047R mutations [59 (link)]. It was kindly provided by Prof. James Fagin (Memorial Sloan Kettering Cancer Center), validated by our group by STR profiling (Microsynth, Switzerland), and cultured in DMEM medium 10% FCS, 2 mM L-glutamine, MEM NEAA (Thermo Fisher) 1:100 and P/S (100U Penicilin/ml and 0.1mg Streptomycin/ml). The mutational status for PIK3CA of all three cell lines was validated by sequencing. All cell lines were cultured for a maximum of 40 passages or 6 months; whichever limit was reached first.

Roelli M.A., Ruffieux-Daidié D., Stooss A., ElMokh O., Phillips W.A., Dettmer M.S, & Charles R.P. (2017). PIK3CAH1047R-induced paradoxical ERK activation results in resistance to BRAFV600E specific inhibitors in BRAFV600E PIK3CAH1047R double mutant thyroid tumors. Oncotarget, 8(61), 103207-103222.

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Cell Line Cultivation and Authentication

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All cell lines, including SK-N-SH, SK-N-BE(2 (link)), SH-SY5Y, SK-N-AS and IMR32, were purchased from Cobioer Biosciences Co., Ltd. The SK-N-SH and IMR32 cells were cultured in minimum essential medium (MEM; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% 1 mM sodium pyruvate (Gibco; Thermo Fisher Scientific, Inc.) and 1% MEM non-essential amino acids (MEM NEAA; Gibco; Thermo Fisher Scientific, Inc.). The SK-N-BE(2 (link)) and SH-SY5Y cells were cultured in MEM/F12 (1:1) (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS, 1% 1 mM sodium pyruvate and 1% MEM NEAA. The SK-N-AS cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS. All culture mediums were supplemented with 1% penicillin-streptomycin solution (Gibco; Thermo Fisher Scientific, Inc.). All cells were cultured in a 5% CO₂ and humidified incubator maintained at 37°C. All cell lines had been authenticated by STR profiling.

Li M., Hu Y., Wang J., Xu Y., Hong Y., Zhang L., Luo Q., Zhen Z., Lu S., Huang J., Zhu J., Zhang Y., Que Y, & Sun F. (2023). The dual HDAC and PI3K inhibitor, CUDC-907, inhibits tumor growth and stem-like properties by suppressing PTX3 in neuroblastoma. International Journal of Oncology, 64(2), 14.

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Adipogenic Differentiation of Porcine AD-MSCs

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Mesenchymal stem cells were isolated from adipose tissue (AD-MSC) of a three-month-old Large White pig, as described earlier^{68 (link)}. Cells were cultured in Advanced DMEM supplemented with 10% FBS, 5 ng/ml FGF-2 (PromoKine), 2 mM l-Glutamine (PAA), 1 mM 2-mercaptoethanol (Sigma), 1 × antibiotic antimycotic solution (Sigma) and 1 × MEM NEAA (Thermo Fisher) at 37 °C in 5% CO₂. Adipogenesis was induced when the cells reached confluency by culturing cells in adipogenic medium, which consisted of Advanced DMEM (Gibco), 10% FBS (Sigma), 1 × antibiotic antimycotic solution (Sigma), 1 × MEM NEAA (Thermo Fisher), 5 ng/ml FGF-2 (PromoKine), 1 × Linoleic Acid Albumin , 1 × ITS, 1 µm Dexamethasone (Sigma), 100 µm Indomethacin (Sigma) and 50 mM IBMX (Sigma). The adipogenic differentiation lasted for seven days and was monitored under a phase-contrast microscope (TS100 Eclipse, Nikon).

Stachecka J., Kolodziejski P.A., Noak M, & Szczerbal I. (2021). Alteration of active and repressive histone marks during adipogenic differentiation of porcine mesenchymal stem cells. Scientific Reports, 11, 1325.

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Establishment and Culture of Murine PDAC Cell Lines

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The KPC cell line is an established PDAC tumor cell line originally derived from transgenic KPC mice harboring tissue-specific mutations in Kras and p53 through expression of cre recombinase under control of pancreatic-specific pdx-1 promoter. The KPC mice were previously backcrossed to the C57Bl/6 mouse background for nine generations as described[29 (link)]. Cells were grown in KPC media containing RPMI 1640 medium (Life Technologies) supplemented with 10% heat inactivated FBS (Benchmark), 2mM L-glutamine (Life Technologies), 1% MEM-NEAA (Life Technologies), 1 mM sodium pyruvate (Sigma Aldrich), and 100 units/mL penicillin and 100 μg/mL streptomycin (Life Technologies). KPC cells were maintained in culture at 37°C in 5% CO₂. B78H1 cells are a murine fibroblast cell line engineered to secrete GM-CSF. B78H1 cells were grown in B78H1 media containing RPMI 1640 medium supplemented with 10% heat inactivated FBS, 1mM L-glutamine, and 100 units/mL penicillin and 100 μg/mL streptomycin. B78H1 cells were maintained in culture at 37°C in 5% CO₂.
Harvested tumor-infiltrating immune cells were processed in CTL media consisting of RPMI 1640 medium, 10% heat inactivated FBS, 1% MEM-NEAA, 1mM L-glutamine, 1% HEPES (Life Technologies), 100 units/mL penicillin and 100 μg/mL streptomycin, and 50 μM 2-mercaptoethanol (Sigma).

Muth S.T., Saung M.T., Blair A.B., Henderson M.G., Thomas DL I.I, & Zheng L. (2020). CD137 Agonist-based Combination Immunotherapy Enhances Activated, Effector Memory T cells and Prolongs Survival in Pancreatic Adenocarcinoma. Cancer letters, 499, 99-108.

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Feeder-Free Culture of Human Pluripotent Stem Cells

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For gene targeting, H1 human pluripotent stem cells²⁸ (link) were maintained in DMEM-F12 (Life Technologies cat# 11330-057) supplemented with 20% KSR (Life Technologies cat# 10828-028), 4 ng/mL FGF (Life Technologies cat# PHG0261), 0.1 mM 2-mercaptoethanol (Life Technologies cat #21985-023), 1x L-glutamine (Life Technologies cat# 25030-081), 1x MEM-NEAA (Life Technologies cat# 11140-050), 1x penicillin/streptomycin (Life Technologies cat# 15140-122). Cells were passaged with 1 mg/mL collagenase IV (Life Technologies cat# 17104019) every 4-6 days on mitomycin C inactivated MEF feeders cultured in DMEM medium supplemented with 10% FBS, 1x MEM-NEAA, 1x penicillin/streptomycin, and 1x L-glutamine. For differentiation to β-like cells, H1 cells and targeted H1 cells were adapted for feeder-free conditions by culturing in Matrigel (Corning) coated tissue culture plates in mTeSR (Stem Cell Technology). For coating plates, Matrigel (Corning) was 1:100 diluted in cold DMEM-F12 medium, and 2 mL of diluted Matrigel solution was added to one well of a 6-well plate well, and coat for overnight at room temperature before pre-warming in a 37°C incubator for 1 hour before using. Cells were cultured in mTeSR medium in 6-well plates and passaged very 3-4 days with a 1:4-6 passage ratio.

Ma H., Jeppesen J.F, & Jaenisch R. (2020). Human T Cells Expressing a CD19 CAR-T Receptor Provide Insights into Mechanisms of Human CD19-Positive β Cell Destruction. Cell Reports Medicine, 1(6), 100097.

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Differentiation of Neurogenin 2-Induced Human Neurons

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Neurogenin 2 (Ngn2)-induced human neurons (Zhang et al., 2013 (link)) were prepared as described in Figure 1A (Hong et al., 2018 (link); Jin et al., 2018 (link)). In brief, YZ1 iPSCs were maintained in medium containing DMEM/F12, Knockout Serum Replacement, penicillin/streptomycin, L-glutamine, MEM-NEAA, and β-mercaptoethanol (all from Invitrogen, Carlsbad, CA, USA) with addition of 10 μg/mL basic fibroblast growth factor (bFGF; Millipore-Sigma, Burlington, MA, USA). iPSCs were plated at a density of 95,000 cells/cm² for viral infection. Ultrapure lentiviral titers were obtained from Alstem (Richmond, CA, USA) and used at the following concentrations: Tet-O-Ngn2-puro: 0.1 μL/50,000 cells; Tet-O-FUW-EGFP: 0.05 μL/50,000 cells; FUdeltaGW-rtTA: 0.11 μL/50,000 cells. To induce Ngn-2 expression, doxycycline was added on iN day 1 at a concentration of 2 μg/mL. On iN day 2, puromycin was added at 20 μg/mL and was maintained in medium thereafter. On iN day 4, cells were plated on Matrigel (Corning, NY)-coated 96-well plates (5,000 cells/well) and maintained in Neurobasal media (Gibco, Carlsbad, CA, USA) containing Glutamax, 20% dextrose, MEM-NEAA, B27 and 10 ng/mL BDNF, CNTF, GDNF (PeproTech, Rocky Hill, NY, USA). On iN day 21, cells were used to investigate effects of AD brain extracts +/− mAbs.

Mengel D., Hong W., Corbett G.T., Liu W., DeSousa A., Solforosi L., Fang C., Frosch M.P., Collinge J., Harris D, & Walsh D.M. (2018). PrP-grafted antibodies bind certain amyloid β-protein aggregates, but do not prevent toxicity. Brain research, 1710, 125-135.

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Cortical Organoid Differentiation Protocol

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Cortical organoids initiate from EBs which are 3D hiPSC-derived multicellular clusters. To form EBs, single hiPSCs were resuspended in mTeSR1 medium containing the ROCK inhibitor Y27632 at 15 μM, seeded on micropillar chips, and maintained for 1 day. On the 2nd day, the medium was replaced with KSR medium supplemented with additional factors. Knockout serum replacement (KSR) medium included the following ingredients: 20% KSR (Invitrogen), 80% DMEM/F12 medium (Invitrogen), 1% GlutaMAX (Invitrogen), 1% minimum essential media-nonessential amino acids (MEM-NEAA, Invitrogen), penicillin–streptomycin (Sigma), 0.2 mM 2-mercaptoethanol (Sigma-Aldrich), the ROCK inhibitor Y27632 at 15 μM and 4 ng ml⁻¹ basic fibroblast growth factor (bFGF, Peprotech). In addition to the reagents above, the medium also contained two inhibitors, the AMPK inhibitor dorsomorphin (Selleck) and the TGF-β inhibitor A83–01 (Sigma). dorsomorphin, A83–01 and Y27632 were added to the KSR medium for the first 3 days. From day 4 to day 6, the KSR medium contained dorsomorphin/A83–01 and bFGF. On day 6, the KSR medium was replaced with neural induction medium (NIM) consisting of DMEM/F12, 1% MEM-NEAA, 1% N2 supplement (Invitrogen), 1% penicillin–streptomycin, 1% GlutaMAX, and 1 μg ml⁻¹ heparin (Sigma). From day 6 to day 9, the NIM contained bFGF. From day 9 to day 11, the cortical organoids were cultured in NIM.

Cui K., Wang Y., Zhu Y., Tao T., Yin F., Guo Y., Liu H., Li F., Wang P., Chen Y, & Qin J. (2020). Neurodevelopmental impairment induced by prenatal valproic acid exposure shown with the human cortical organoid-on-a-chip model. Microsystems & Nanoengineering, 6, 49.

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Cell Line Characterization and Maintenance

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HEK-293T (293T), FaDu, UPCI:SCC154 (SCC154) and UPCI:SCC152 (SCC152) cell lines were obtained from the American Type Culture Collection (ATCC). HN5 and UMSCC47 cell lines were obtained from Dr. Jeffery Myers, UT MD Anderson Cancer Center, Houston, TX, USA. FaDu, UPCI:SCC154 and UPCI:SCC152 cells were maintained in MEM (Gibco) supplemented with 10% heat inactivated Fetal Bovine Serum (Sigma), 1% MEM NEAA (Gibco), Sodium Pyruvate (Gibco) and Penicillin Streptomycin Solution (Corning). UMSCC47 cells were maintained in DMEM (Gibco) supplemented with 10% heat inactivated Fetal Bovine Serum (Sigma), 2% MEM Vitamin Solution (Gibco), 1% MEM NEAA (Gibco), Sodium Pyruvate (Gibco) and Penicillin Streptomycin Solution (Corning). 293T and HN5 cells were maintained in DMEM/F-12 50/50 (Corning) supplemented with 10% heat inactivated Fetal Bovine Serum (Sigma) and 1% Penicillin Streptomycin Solution (Corning). All cell lines used were authenticated by STR fingerprinting by the Characterized Cell Line Core Facility at UT MD Anderson.

Molkentine J.M., Molkentine D.P., Bridges K.A., Xie T., Yang L., Sheth A., Heffernan T.P., Clump D.A., Faust A.Z., Ferris R., Myers J.N., Frederick M.J., Mason K.A., Meyn R.E., Pickering C.R, & Skinner H.D. (2020). Targeting DNA damage response in head and neck cancers through abrogation of cell cycle checkpoints. International journal of radiation biology, 97(8), 1121-1128.

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