Glutamax 100x

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom, Spain

GlutaMAX (100X) is a stable glutamine substitute for cell culture media. It provides a consistent source of L-glutamine to support cell growth and proliferation.

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34 protocols using glutamax 100x

Cell Culture and Transfection Protocols

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HEK293 (XX female) and COS7 (XY male) cells were cultured in high glucose Dulbecco’s modified eagle medium (DMEM, GIBCO, 41965) supplemented with 10% fetal bovine serum (FBS, GIBCO, 10270, South America origin), 1% GlutaMAX (100X) (GIBCO, 35050), and 100 units/mL penicillin and 100 μg/mL streptomycin (Sigma-Aldrich, P0781). U87MG (XY male) cells were cultured in MEM with non-essential amino acids (Sigma) supplemented with 10% FBS (GIBCO), 1mM Sodium pyruvate, 1% GlutaMAX (100X) (GIBCO, 35050), and 100 units/mL penicillin and 100 μg/mL streptomycin (Sigma-Aldrich, P0781). HUVECs (XX, female) cells were cultured in M199 (with Glutamine) (Sigma-Aldrich, M4530), supplemented with 20% FBS (GIBCO), 1% GlutaMAX (100X) (GIBCO, 35050), 100 units/mL penicillin and 100 μg/mL streptomycin (Sigma-Aldrich, P0781), 1% Vitamins- MEM-EAGLE Vitamin Solution (GIBCO, 11120052). HUVEC cells were grown in flasks coated with gelatin (Sigma-Aldrich, G1890). U87MG and HUVEC cells were a kind gift from Gera Neufeld (the Rappaport Institute). Cells were incubated at 37°C in humidified air containing 5% CO₂ and transfections were performed using Lipofectamine 2000 (GIBCO, 11668) in serum-free Opti-MEM I medium (GIBCO, 31985) according to the manufacturer’s protocols.

van der Klaauw A.A., Croizier S., Mendes de Oliveira E., Stadler L.K., Park S., Kong Y., Banton M.C., Tandon P., Hendricks A.E., Keogh J.M., Riley S.E., Papadia S., Henning E., Bounds R., Bochukova E.G., Mistry V., O’Rahilly S., Simerly R.B., Minchin J.E., Barroso I., Jones E.Y., Bouret S.G, & Farooqi I.S. (2019). Human Semaphorin 3 Variants Link Melanocortin Circuit Development and Energy Balance. Cell, 176(4), 729-742.e18.

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Primary Neuronal Culture with α-Syn Exposure

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Primary neuronal culture was performed as described previously (10 (link),21 (link),22 (link)). Freshly dissociated (trypsin) hippocampi were plated (10⁵ cells/well in a 12-well dish containing an 18-mm coverslip) in neuronal attachment media consisting of 10% horse serum (Eurobio), 1 mM sodium pyruvate (Thermo Fisher Scientific), 2 mM Glutamax-100X (Thermo Fisher Scientific), and penicillin/streptomycin (Thermo Fisher Scientific) in MEM (Thermo Fisher Scientific) for 3 h. The attachment medium was replaced, and cells were maintained in serum-free neurobasal medium (Thermo Fisher Scientific) supplemented with B27 (Gibco, Gaithersburg, MD) and 2 mM Glutamax-100X. Exposure to fibrillar α-Syn polymorphs was performed at days in vitro 14 (DIV 14). Fibrillar α-Syn polymorphs were diluted in fresh neurobasal medium. The “cell-conditioned neurobasal medium” was replaced with fibrillar α-Syn containing neurobasal medium for 15 min, the former kept aside at 37°C. After 15 min exposure, fibrillar-α-Syn-containing medium was removed, and the well was washed thrice. Lastly, the cells were replenished with “cell-conditioned neurobasal medium” and transferred back to the incubator until DIV 21.

Shrivastava A.N., Bousset L., Renner M., Redeker V., Savistchenko J., Triller A, & Melki R. (2020). Differential Membrane Binding and Seeding of Distinct α-Synuclein Fibrillar Polymorphs. Biophysical Journal, 118(6), 1301-1320.

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Preparation of Primary Mouse Neuronal Cultures

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Primary neurons were prepared from embryonic day 18 C57BL/6J mice (Janvier Labs, France). For biochemistry experiments, (proteomics studies and cell‐surface biotinylation), cortical neuronal cultures were plated on 6‐cm plate pre‐coated with 80 mg/ml poly‐D, L‐ornithine (Sigma, 24–48 h; Shrivastava et al, 2013b, 2015). Cortical neurons were used, as they can be prepared in larger quantities (2 × 10⁶ cells/dish) as required for these experiments. All other experiments were performed on hippocampal neuronal cultures plated on 18 mm coverslips pre‐coated (24–48 h) with 80 mg/ml poly‐D, L‐ornithine. Freshly dissociated (trypsin) hippocampal cells were plated (1 × 10⁵ cells/well) in neuronal attachment media consisting of 10% horse serum (Eurobio), 1 mM sodium pyruvate (GIBCO), 2 mM Glutamax‐100X (GIBCO), and penicillin/streptomycin (GIBCO) in MEM (GIBCO) for 3 h. The attachment medium was replaced and cells were maintained in serum‐free neurobasal medium (GIBCO) supplemented with 1X B27 (GIBCO) and 2 mM Glutamax‐100X. Cells were maintained by supplementing with fresh serum‐free neurobasal medium every week.

Shrivastava A.N., Redeker V., Pieri L., Bousset L., Renner M., Madiona K., Mailhes‐Hamon C., Coens A., Buée L., Hantraye P., Triller A, & Melki R. (2019). Clustering of Tau fibrils impairs the synaptic composition of α3‐Na+/K+‐ATPase and AMPA receptors. The EMBO Journal, 38(3), e99871.

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Isolation and Culture of Murine Spinal Cord Glial Cells

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Primary cultures of glial cells were established from the spinal cord of 16-day-old C57/Bl6 mice (Harlan). Animals were sacrificed in aseptic conditions. Spinal cords were dissected, freed from meninges and collected in cold HBSS supplemented with calcium and magnesium (Gibco), glucose (Sigma, 6 g/L) and 1% antibiotic solution (penicillin/streptomycin, Gibco). Tissues were chopped and rinsed (3 times) in HBSS (without calcium and magnesium, 1% antibiotic solution), supernatant was removed. Tissues were re-suspended in 1.5 ml of DMEM/F12 medium (Invitrogen) and 1% penicillin/streptomycin and treated with 2 ml 0.25% trypsin-EDTA (Gibco) for 20 minutes at 37°C. Trypsin was inactivated by adding 10% FBS. DNAse1 (10mg/ml, Rotkreuz. Switzerland) was then added. Cells were mechanically dissociated and re-suspended in 10ml of culture medium consisting of DMEM/F12, glucose 6 g/L, glutamax 100X (Gibco), 10% FBS (Gibco) and 1% penicillin/streptomycin. Centrifugation (5min, 500g, room temperature) was done and supernatant removed. Cells were re-suspended in the same culture medium and plated at a final concentration of 50 000 cells/well on glass coverslips treated with 25 μg/ml of low-molecular weight poly-D-lysine (Sigma-Aldrich, St Louis, MO) in 24-well dishes (Nunc, Roskilde, Danemark).

Salinas S., Constant O., Desmetz C., Barthelemy J., Lemaitre J.M., Milhavet O., Nagot N., Foulongne V., Perrin F.E., Saiz J.C., Lecollinet S., Van de Perre P, & Simonin Y. (2017). Deleterious effect of Usutu virus on human neural cells. PLoS Neglected Tropical Diseases, 11(9), e0005913.

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HEK293 and HeLa Cell Culture

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HEK293 (XX female) and suspension HeLa (XX female) cells were cultured in high glucose Dulbecco’s modified eagle medium (DMEM, GIBCO, 41965) and supplemented with 10% fetal bovine serum (GIBCO, 10270, South America origin), 1% GlutaMAX (100X) (GIBCO, 35050), and 100 units/mL penicillin and 100 μg/mL streptomycin (Sigma-Aldrich, P0781). Cells were incubated at 37°C in humidified air containing 5% CO₂ and transfections were performed using Lipofectamine 2000 (GIBCO, 11668) in serum-free Opti-MEM I medium (GIBCO, 31985) according to the manufacturer’s protocols.

Lotta L.A., Mokrosiński J., Mendes de Oliveira E., Li C., Sharp S.J., Luan J., Brouwers B., Ayinampudi V., Bowker N., Kerrison N., Kaimakis V., Hoult D., Stewart I.D., Wheeler E., Day F.R., Perry J.R., Langenberg C., Wareham N.J, & Farooqi I.S. (2019). Human Gain-of-Function MC4R Variants Show Signaling Bias and Protect against Obesity. Cell, 177(3), 597-607.e9.

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Cell Culture Conditions for HEK293 and COS-7 Cells

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HEK293 (XX female) cells were cultured in high glucose Dulbecco’s modified eagle medium (DMEM, GIBCO, 41965) supplemented with 10% fetal bovine serum (GIBCO, 10270, South America origin), 1% GlutaMAX (100X) (GIBCO, 35050), and 100 units/mL penicillin and 100 μg/mL streptomycin (Sigma-Aldrich, P0781) at 37°C, 5% CO2. COS-7 (XY male) cells were cultured in low glucose Dulbecco’s modified Eagle’s medium (Sigma, D6046) supplemented with 10% fetal bovine serum, 1% GlutaMAX, 100 IU/mL penicillin and 100 ng/mL streptomycin at 37°C, 5% CO2.

Marenne G., Hendricks A.E., Perdikari A., Bounds R., Payne F., Keogh J.M., Lelliott C.J., Henning E., Pathan S., Ashford S., Bochukova E.G., Mistry V., Daly A., Hayward C., Wareham N.J., O’Rahilly S., Langenberg C., Wheeler E., Zeggini E., Farooqi I.S, & Barroso I. (2020). Exome Sequencing Identifies Genes and Gene Sets Contributing to Severe Childhood Obesity, Linking PHIP Variants to Repressed POMC Transcription. Cell Metabolism, 31(6), 1107-1119.e12.

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Investigating GPR83 and MC3R Signaling

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To investigate the effect of GPR83 of MC3R signalling, we performed in vitro assays in transiently transfected HEK293 cells maintained in Dulbecco’s modified eagle medium (high glucose DMEM, GIBCO, 41965) supplemented with 10% fetal bovine serum (GIBCO, 10270), 1% GlutaMAX^™ (100X) (GIBCO, 35050), and 100 units/mL penicillin and 100 mg/mL streptomycin (Sigma-Aldrich, P0781). Cells were incubated at 37°C in humidified air containing 5% CO2 and transfections were performed using Lipofectamine 2000 (GIBCO, 11668) in serum-free Opti-MEM I medium (GIBCO, 31985), according to the manufacturer’s protocols. The plasmids used encode the C-FLAG-tagged human GPR83 WT (NM_016540.4) or N-FLAG-tagged human MC3R WT (NM_019888.3) ligated into pcDNA3.1(+) (Invitrogen).

Kentistou K.A., Kaisinger L.R., Stankovic S., Vaudel M., de Oliveira E.M., Messina A., Walters R.G., Liu X., Busch A.S., Helgason H., Thompson D.J., Santon F., Petricek K.M., Zouaghi Y., Huang-Doran I., Gudbjartsson D.F., Bratland E., Lin K., Gardner E.J., Zhao Y., Jia R., Terao C., Riggan M., Bolla M.K., Yazdanpanah M., Yazdanpanah N., Bradfield J.P., Broer L., Campbell A., Chasman D.I., Cousminer D.L., Franceschini N., Franke L.H., Girotto G., He C., Järvelin M.R., Joshi P.K., Kamatani Y., Karlsson R., Luan J., Lunetta K.L., Mägi R., Mangino M., Medland S.E., Meisinger C., Noordam R., Nutile T., Concas M.P., Polašek O., Porcu E., Ring S.M., Sala C., Smith A.V., Tanaka T., van der Most P.J., Vitart V., Wang C.A., Willemsen G., Zygmunt M., Ahearn T.U., Andrulis I.L., Anton-Culver H., Antoniou A.C., Auer P.L., Barnes C.L., Beckmann M.W., Berrington A., Bogdanova N.V., Bojesen S.E., Brenner H., Buring J.E., Canzian F., Chang-Claude J., Couch F.J., Cox A., Crisponi L., Czene K., Daly M.B., Demerath E.W., Dennis J., Devilee P., Vivo I.D., Dörk T., Dunning A.M., Dwek M., Eriksson J.G., Fasching P.A., Fernandez-Rhodes L., Ferreli L., Fletcher O., Gago-Dominguez M., García-Closas M., García-Sáenz J.A., González-Neira A., Grallert H., Guénel P., Haiman C.A., Hall P., Hamann U., Hakonarson H., Hart R.J., Hickey M., Hooning M.J., Hoppe R., Hopper J.L., Hottenga J.J., Hu F.B., Hübner H., Hunter D.J., Jernström H., John E.M., Karasik D., Khusnutdinova E.K., Kristensen V.N., Lacey J.V., Lambrechts D., Launer L.J., Lind P.A., Lindblom A., Magnusson P.K., Mannermaa A., McCarthy M.I., Meitinger T., Menni C., Michailidou K., Millwood I.Y., Milne R.L., Montgomery G.W., Nevanlinna H., Nolte I.M., Nyholt D.R., Obi N., O’Brien K.M., Offit K., Oldehinkel A.J., Ostrowski S.R., Palotie A., Pedersen O.B., Peters A., Pianigiani G., Plaseska-Karanfilska D., Pouta A., Pozarickij A., Radice P., Rennert G., Rosendaal F.R., Ruggiero D., Saloustros E., Sandler D.P., Schipf S., Schmidt C.O., Schmidt M.K., Small K., Spedicati B., Stampfer M., Stone J., Tamimi R.M., Teras L.R., Tikkanen E., Turman C., Vachon C.M., Wang Q., Winqvist R., Wolk A., Zemel B.S., Zheng W., van Dijk K.W., Alizadeh B.Z., Bandinelli S., Boerwinkle E., Boomsma D.I., Ciullo M., Chenevix-Trench G., Cucca F., Esko T., Gieger C., Grant S.F., Gudnason V., Hayward C., Kolčić I., Kraft P., Lawlor D.A., Martin N.G., Nøhr E.A., Pedersen N.L., Pennell C.E., Ridker P.M., Robino A., Snieder H., Sovio U., Spector T.D., Stöckl D., Sudlow C., Timpson N.J., Toniolo D., Uitterlinden A., Ulivi S., Völzke H., Wareham N.J., Widen E., Wilson J.F., Pharoah P.D., Li L., Easton D.F., Njølstad P., Sulem P., Murabito J.M., Murray A., Manousaki D., Juul A., Erikstrup C., Stefansson K., Horikoshi M., Chen Z., Farooqi I.S., Pitteloud N., Johansson S., Day F.R., Perry J.R, & Ong K.K. (2023). Understanding the genetic complexity of puberty timing across the allele frequency spectrum. medRxiv.

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Establishing Primary Sarcoma Cell Cultures

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We successfully generated 5 primary sarcoma cell cultures, 2 OS and 3 UPS, starting from biopsies or thoracentesis of metastatic (n = 4) or primitive (n = 1) tumor sites. All individuals provided informed consent according to a protocol approved by the Internal Review Board and Ethic Committee.
Four sarcoma cultures (S1, S3, S5 and S16) were previously obtained from surgical biopsy and characterized by our group.³⁰ S22 osteosarcoma culture was generated starting from freshly isolated thoracentesis, obtained in a sterile vacuum bottle. Tumoral cells were isolated from fluid by centrifugation and resuspended in KnockOut Dulbecco’s Modified Eagle’s: Nutrient Mixture F-12 Medium (KO DMEM:F12 medium, Gibco BRL, Life Technologies Italia) with the addition of penicillin (50 U/ml), streptomycin (50 μg/ml), Glutamax 100X (all from Gibco BRL, Life Technologies Italia). Cells were seeded in 10% heat-inactivated Fetal Bovine Serum (FBS, Euroclone Spa), in multi-well plates treated for anchorage-dependent cultures (Corning/Costar, VWR International PBI S.r.l.) at clonal density (10⁴-10⁵ cells per cm²).

Mesiano G., Grignani G., Fiorino E., Leuci V., Rotolo R., D’Ambrosio L., Salfi C., Gammaitoni L., Giraudo L., Pisacane A., Butera S., Pignochino Y., Basiricó M., Capozzi F., Sapino A., Aglietta M, & Sangiolo D. (2018). Cytokine Induced Killer cells are effective against sarcoma cancer stem cells spared by chemotherapy and target therapy.. Oncoimmunology, 7(11), e1465161.

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Fibroblast Staining Protocol for GLB1 Deficiency

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Fibroblasts were cultured in MEM (Gibco) supplemented with 15% FBS (Atlanta Biologicals), Glutamax 100x (Gibco) and penicillin/streptomycin 100x (Sigma). Normal human fibroblasts (GLB1^+/+; Coriell GM-00010) and GM1 fibroblasts (GLB1^−/−; GM-10919) were grown in 35 mm glass bottom dishes (MatTek) for 24 h. The media was then replaced in the GM1 fibroblasts with serum-free MEM containing 500 ng/ml of β-gal:RTB. The media was also replaced with serum-free MEM in normal and GM1 fibroblasts as controls. Samples were incubated for 2 h at 37°C to allow uptake of β-gal:RTB. Cells were then fixed with 0.1 M phosphate buffer, pH 7.3 supplemented with 5 mM EGTA (Sigma), 2 mM MgCl₂ and 0.2% glutaraldehyde (Sigma) for 15 min at room temperature. Cells were subsequently washed 2X with wash buffer (5 min each; 0.1 M phosphate buffer, 2 mM MgCl₂ pH 7.3). X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) at 1 mg/ml was prepared in staining buffer [0.1 M citrate phosphate buffer, pH 4.6, 5 mM K₄Fe(CN)₆-3H₂0 (Sigma) and 5 mM K₃Fe(CN)₆ (Sigma)] and filtered through a 0.2 um filter to remove crystals. X-gal solution was added to the fibroblasts and incubated overnight inside a humidified chamber at 37°C. X-gal solution was removed; fibroblasts were washed 3X with wash buffer, and images were acquired using a Nikon TE2000 phase contrast microscope with a 60X objective.

Condori J., Acosta W., Ayala J., Katta V., Flory A., Martin R., Radin J., Cramer C.L, & Radin D.N. (2015). Enzyme replacement for GM1-gangliosidosis: Uptake, lysosomal activation, and cellular disease correction using a novel β-galactosidase:RTB lectin fusion. Molecular genetics and metabolism, 117(2), 199-209.

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Mitochondrial Protein Synthesis Labeling

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Cells on a six-well plate were washed twice for 5 min each in methionine/cysteine-free DMEM and incubated with methionine/cysteine-free DMEM supplemented with 10% dialyzed FBS, GlutaMax 100X (Gibco), sodium pyruvate 100× (Gibco) and 100 μg/ml emetine (Sigma-Aldrich) (an irreversible cytosolic translation inhibitor) for 20 min at 37°C. Cells were then incubated in the same media supplemented with 166.7 μCi/ml of [³⁵S]-methionine (Perkin Elmer) for 30 min at 37°C to label the newly synthesized mitochondrially encoded proteins. After washing three times with PBS, cells were lysed and 30 μg aliquots were separated on 10–20% Tris-glycine (Invitrogen) SDS-PAGE gels. Gels were dried for 2 h at 65°C and exposed on a phosphoimager screen prior to visualization with Typhoon FLA 7000 phosphoimager.

Gopalakrishna S., Pearce S.F., Dinan A.M., Rosenberger F.A., Cipullo M., Spåhr H., Khawaja A., Maffezzini C., Freyer C., Wredenberg A., Atanassov I., Firth A.E, & Rorbach J. (2019). C6orf203 is an RNA-binding protein involved in mitochondrial protein synthesis. Nucleic Acids Research, 47(17), 9386-9399.

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