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Mem nea

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The MEM NEA is a laboratory equipment product from Thermo Fisher Scientific. It is designed to provide a consistent and reliable environment for cell culture applications. The core function of the MEM NEA is to maintain the optimal pH, temperature, and gas composition required for the growth and maintenance of cells in vitro.

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Lab products found in correlation

Penicillin streptomycin, by Thermo Fisher Scientific (7 mentions) Glutamax, by Thermo Fisher Scientific (6 mentions) 2 mercaptoethanol, by Thermo Fisher Scientific (5 mentions) Hepes buffer, by Thermo Fisher Scientific (4 mentions) Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (3 mentions) Fetal bovine serum (fbs), by STEMCELL (3 mentions) Sodium pyruvate, by Thermo Fisher Scientific (2 mentions) Lymphoprep, by STEMCELL (2 mentions) Penicillin, by Thermo Fisher Scientific (2 mentions) Chick serum, by Merck Group (2 mentions) Dispase, by Thermo Fisher Scientific (2 mentions) Flt3l, by Thermo Fisher Scientific (2 mentions) Stem cell factor (scf), by Thermo Fisher Scientific (2 mentions) Low attachment plate, by Corning (2 mentions) Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (2 mentions) L glutamine, by Thermo Fisher Scientific (2 mentions) Trypsin edta, by Thermo Fisher Scientific (2 mentions) Bone morphogenetic protein 4 (bmp4), by R&D Systems (2 mentions) A2780, by Thermo Fisher Scientific (1 mentions) Rpmi 1640, by Thermo Fisher Scientific (1 mentions)

12 protocols using mem nea

1

Ovarian Cancer Cell Culture Protocol

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PA1 (Ovarian Teratocarcinoma origin) and A2780 (Ovarian epithelial origin) cell lines were purchased from NCCS (Pune, India) and ATCC (Manassas, VA, USA) respectively. PA1 cells were cultured in MEM (GIBCO, Lonza) containing 10% FBS (GIBCO, South America origin), 1X Antibiotic-Antimycotic (GIBCO) and 1X MEM NEA (GIBCO); A2780 cell lines were cultured in RPMI-1640 supplemented with 10% FBS (GIBCO, South America origin) and 1X Antibiotic-Antimycotic (GIBCO); all the cell lines were maintained at 37°C with 5% CO2 in a humidified incubator.
Koroth J., Mahadeva R., Ravindran F., Parashar T.R., Teja V., Karki S.S, & Choudhary B. (2021). Curcumin derivative 1, 2-bis [(3E, 5E)-3, 5-bis [(2-chlorophenyl) methylene]-4-oxo-1-piperidyl] ethane-1, 2-dione (ST03) induces mitochondria mediated apoptosis in ovarian cancer cells and inhibits tumor progression in EAC mouse model. Translational Oncology, 15(1), 101280.
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2

Isolation of PBMCs from Blood

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PBMCs were isolated by Ficoll density gradient centrifugation. The gradient was a commercially available Ficoll separation medium (Lymphoprep, Stem Cell Technologies, Cologne, Germany). PBMCs were resuspended in RPMI 1640 medium with Glutamax (Gibco Life Technologies, Darmstadt, Germany) supplemented with 10% heat-inactivated fetal calf serum (Greiner Bio-One, Frickenhausen, Germany), of penicillin (100 U/ml) and streptomycin (1 µg/ml) as well as non-essential amino acids (NEAs) (diluted according to manufacturer’s instructions, MEM NEA) and sodium pyruvate (1 mM, all from Gibco Life Technologies, Schwerte, Germany).
Brinkhoff A., Zeng Y., Sieberichs A., Dolff S., Shilei X., Sun M., Engler H., Benson S., Korth J., Schedlowski M., Kribben A., Witzke O, & Wilde B. (2019). B-cell dynamics during experimental endotoxemia in humans. Bioscience Reports, 39(5), BSR20182347.
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3

PBMC Isolation via Ficoll Gradient

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PBMC isolation was facilitated by Ficoll density gradient centrifugation. The gradient was a commercially available Ficoll separation medium (Lymphoprep, STEMCELL Technologies, Köln, Germany). PBMCs were resuspended in RPMI 1640 medium with Glutamax (Gibco Life Technologies, Darmstadt, Germany) supplemented with 10% heat-inactivated fetal calf serum (Greiner Bio-One, Frickenhausen, Germany), 2% of penicillin and streptomycin, as well as with non-essential amino acids (MEM NEA) and sodium pyruvate (all from Gibco Life Technologies).
Brinkhoff A., Sieberichs A., Engler H., Dolff S., Benson S., Korth J., Schedlowski M., Kribben A., Witzke O, & Wilde B. (2018). Pro-Inflammatory Th1 and Th17 Cells Are Suppressed During Human Experimental Endotoxemia Whereas Anti-Inflammatory IL-10 Producing T-Cells Are Unaffected. Frontiers in Immunology, 9, 1133.
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4

Differentiation and Stimulation of Monocyte-Derived DCs and Macrophages

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Monocytes (>95% pure) were positively selected using the CD14+ monocyte isolation kit (Miltenyi Biotec) and then transferred into 6-well plates (2 × 106 cells per well) in RPMI 1640-Glutamax medium, supplemented with 10% FBS, 100 U ml−1 penicillin, 100 mg ml−1 streptomycin, 1 mM sodium pyruvate, and MEM non-essential amino acids (MEM-NEA) (all from Gibco). DCs were generated by culturing monocytes with recombinant human GM-CSF (50 ng ml−1, Miltenyi Biotec) and IL-4 (10 ng ml−1, R&D Systems), while macrophages were differentiated with recombinant human M-CSF (10 ng ml−1, R&D Systems). On day 3, the cytokine-supplemented medium was refreshed. On day 7, DCs and macrophages were plated into fresh plates of 96 wells (1.5 × 105 per well) or 24 wells (1 × 106 cells per well) in complete medium and stimulated for 5 h with 1 μg ml−1E. coli-derived LPS (serotype O55:B5) either alone or in combination with 1 mM ATP added to the culture for the last 30 min.
Viganò E., Diamond C.E., Spreafico R., Balachander A., Sobota R.M, & Mortellaro A. (2015). Human caspase-4 and caspase-5 regulate the one-step non-canonical inflammasome activation in monocytes. Nature Communications, 6, 8761.
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5

Culturing Mouse Embryonic Fibroblasts

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Mouse embryonic fibroblasts (MEF wt) and OPA1-Knockout fibroblasts were from ATCC (CRL-2991 and CRL-2995 respectively). Cells were cultured in complete Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% Premium Fetal bovine serum (FBS, South Africa S1300; Biowest, Nuallé, France), 1% non-essential amino acids (MEM-NEA), 1% penicillin/Streptomycin (final concentration of 100 U/mL of penicillin and 100 μg/mL of streptomycin), all purchased from Gibco. The cells were grown in a humidified incubator (Forma Steri-Cycle ThermoFisher with HEPA filter, 5% CO2) at 37 °C and maintained with a split ratio of 1:15 at 80% of confluence in T75 flasks (Nunc). Trypsine/EDTA 0.05% from Hyclone was used to detach cell cultures from flasks. Dulbecco Phosphate Buffered Saline without calcium and magnesium (DPBS) from Gibco and Phosphate Buffered Saline 1x (PBS) from Hyclone, were used for washing cells.
Muñoz-Úbeda M., Semenzato M., Franco-Romero A., Junquera E., Aicart E., Scorrano L, & López-Montero I. (2021). Transgene expression in mice of the Opa1 mitochondrial transmembrane protein through bicontinuous cubic lipoplexes containing gemini imidazolium surfactants. Journal of Nanobiotechnology, 19, 425.
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6

Embryoid Body Differentiation from hESCs

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hESCs were treated with 0.5 mg/ml Dispase (Invitrogen) in DMEM/F12 for 5 minutes at 37°C. Clumps were mechanically detached and transferred to low attachment plates (Corning) in IMDM (Invitrogen), 15% FBS (Hyclone), 1% MEM-NEA, 0.3 μM BME (Invitrogen) and, 1% penicillin/streptomycin (100 U/ml, Invitrogen)/1mM L-glutamine (P/S/G). Medium was changed every 2 days and supplemented with 10 ng/ml BMP-4 (R&D Systems), 50 ng/ml FLT-3L (Peprotech) and 300 ng/ml SCF (Peprotech) at day 4 of EB differentiation. BMP-4 was removed from the culture medium at day 12. EBs were harvested at 2 weeks and dissociated with 0.25% trypsin-EDTA (Invitrogen) with 2% chick serum (Sigma-Aldrich) for 30 minutes at 37°C with periodic agitation and filtered with 70 μm cell strainer. A step-by-step protocol detailing EB generation and AM580/ATRA treatment can be found at Nature Protocol Exchange57 .
Dou D.R., Calvanese V., Sierra M.I., Nguyen A.T., Minasian A., Saarikoski P., Sasidharan R., Ramirez C.M., Zack J.A., Crooks G.M., Galic Z, & Mikkola H.K. (2016). Medial HOXA genes demarcate haematopoietic stem cell fate during human development. Nature cell biology, 18(6), 595-606.
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7

Conditional CDC6 Overexpression in mESCs

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TetO-CDC6 mESCs derived from TetO-CDC6 mice (35 (link)) were cultured on 0.1% gelatin-coated plates in Dulbecco's modified Eagle's medium (DMEM) with Ultraglutamine 1 and 4.5 g/l glucose (Lonza) supplemented with 15% FBS (Sigma), 50 U/ml penicillin–50 mg/ml streptomycin (Invitrogen), minimum essential medium non-essential aminoacids (MEM NEA; Invitrogen), 100 μM 2-mercaptoethanol (Invitrogen) and 103 U/ml ESGRO mLIF medium supplement (Millipore). To induce CDC6 overexpression, 1 μg/ml doxycycline (dox, Sigma) was added to the medium for 30 h. When indicated, mESCs were treated with 0.5 μM aphidicolin (Sigma-Aldrich) for 2.5 h to induce mild RS.
Jodkowska K., Pancaldi V., Rigau M., Almeida R., Fernández-Justel J.M., Graña-Castro O., Rodríguez-Acebes S., Rubio-Camarillo M., Carrillo-de Santa Pau E., Pisano D., Al-Shahrour F., Valencia A., Gómez M, & Méndez J. (2022). 3D chromatin connectivity underlies replication origin efficiency in mouse embryonic stem cells. Nucleic Acids Research, 50(21), 12149-12165.
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8

Embryoid Body Differentiation from hESCs

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hESCs were treated with 0.5 mg/ml Dispase (Invitrogen) in DMEM/F12 for 5 minutes at 37°C. Clumps were mechanically detached and transferred to low attachment plates (Corning) in IMDM (Invitrogen), 15% FBS (Hyclone), 1% MEM-NEA, 0.3 μM BME (Invitrogen) and, 1% penicillin/streptomycin (100 U/ml, Invitrogen)/1mM L-glutamine (P/S/G). Medium was changed every 2 days and supplemented with 10 ng/ml BMP-4 (R&D Systems), 50 ng/ml FLT-3L (Peprotech) and 300 ng/ml SCF (Peprotech) at day 4 of EB differentiation. BMP-4 was removed from the culture medium at day 12. EBs were harvested at 2 weeks and dissociated with 0.25% trypsin-EDTA (Invitrogen) with 2% chick serum (Sigma-Aldrich) for 30 minutes at 37°C with periodic agitation and filtered with 70 μm cell strainer. A step-by-step protocol detailing EB generation and AM580/ATRA treatment can be found at Nature Protocol Exchange57 .
Dou D.R., Calvanese V., Sierra M.I., Nguyen A.T., Minasian A., Saarikoski P., Sasidharan R., Ramirez C.M., Zack J.A., Crooks G.M., Galic Z, & Mikkola H.K. (2016). Medial HOXA genes demarcate haematopoietic stem cell fate during human development. Nature cell biology, 18(6), 595-606.
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9

Inducible Knockout of Chromatin Remodelers in Mouse ES Cells

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Mouse ES cells were cultured using standard conditions. ES culture media containing Dulbecco’s Modified Eagle’s Medium (Cat# 10829018; Life Technologies), 15% FBS (Cat# ASM-5007; Applied StemCell), Penicillin-Streptomycin (Cat# 15140122; Life Technologies), Glutamax (Cat# 35050061; Life Technologies), HEPES buffer (Cat# 15630080; Life Technologies), 2-mercaptoethanol (Cat# 21985023; Life Technologies), MEM-NEA (Cat# 11140050; Life Technologies), and LIF supplement40 (link), was replaced daily, and ES cells were passaged every 48 hours. For inducible deletions, Smarca4flox/flox actin–CreER ES cells4 (link) or Arid1aflox/flox actin–CreER ES cells, which previously tested negative for mycoplasma contamination using PCR testing, were respectively plated onto irradiated feeder mouse embryonic fibroblasts, treated with 0.8 uM 4-hydroxytamoxifen (Tam) or ethanol (EtOH) for 48 h, and harvested for further experiments after trypsin dissociation at 72 h. A549 cells were obtained from ATCC (Cat# CCL-185), and directly cultured upon receipt without testing for mycoplasma contamination, using F-12K media supplemented with 10% FBS and Penicillin-Streptomycin.
Stanton B.Z., Hodges C., Calarco J.P., Braun S.M., Ku W.L., Kadoch C., Zhao K, & Crabtree G.R. (2016). SMARCA4 ATPase mutations disrupt direct eviction of PRC1 from chromatin. Nature genetics, 49(2), 282-288.
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10

Culturing HEK 293 and ES Cells

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HEK 293 cells (ATCC) were cultured using standard conditions in media containing: DMEM (Life Technologies), 10% FBS (Applied StemCell), and Penicillin-Streptomycin (Life Technologies).
ES cells were cultured using standard conditions in media containing: DMEM (Life Technologies), 7.5% ES-sure FBS (Applied StemCell), 7.5% KnockOut SR (Life Technologies) Penicillin-Streptomycin (Life Technologies), Glutamax (Life Technologies), HEPES buffer (Life Technologies), 2-mercaptoethanol (Life Technologies), and MEM-NEA (Life Technologies). LIF was replaced daily and ES cells were passaged every 48 h.
For long recruitment experiments (>1 h), cells were treated with 3 nM RAP (Selleckchem) and/or 3 nM FK506 (Abcam) added directly to culture dish and changed daily. For short recruitment experiments (≤1 h), cells were treated with 30 nM RAP and/or FK506 in suspension.
Braun S.M., Kirkland J.G., Chory E.J., Husmann D., Calarco J.P, & Crabtree G.R. (2017). Rapid and reversible epigenome editing by endogenous chromatin regulators. Nature Communications, 8, 560.
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