L glutamate

Manufactured by PerkinElmer

Sourced in Spain

L-glutamate is a chemical compound that serves as a core component in various laboratory equipment and assays. It functions as an important amino acid involved in cellular metabolism and signaling processes. The detailed specifications and intended uses for this product may vary based on the specific application and requirements of the laboratory.

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Lab products found in correlation

L 3h glutamate, by PerkinElmer (5 mentions) L glutamine, by PerkinElmer (1 mentions) 2 nbdg, by Thermo Fisher Scientific (1 mentions) Infinite m1000, by Tecan (1 mentions) Tri carb 2800tr liquid scintillation analyzer, by PerkinElmer (1 mentions) Glutamine, by Lonza (1 mentions) Penicillin, by Lonza (1 mentions) Streptomycin, by Lonza (1 mentions) Fetal bovine serum (fbs), by Lonza (1 mentions) Dulbecco s modified eagle s medium, by Lonza (1 mentions)

6 protocols using l glutamate

Radiolabeled Amino Acid Uptake by E. chaffeensis

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E. chaffeensis (purified as described above) in 200 µl SPK buffer with 2 mM l-glutamine was incubated at 65°C for 15 min or pretreated with 50 µM protamine, 10 mM AC or DHP, or 2 mM histidine on ice for 30 min as described above. Each sample in SPK buffer with 2 mM l-glutamine received 0.2 µCi ³H-labeled l-proline (25 to 55 Ci/mmol; PerkinElmer, Waltham, MA), l-glutamate (40 to 80 Ci/mmol; PerkinElmer), or l-glutamine (30 to 60 Ci/mmol; PerkinElmer) along with 1 µM cold l-proline, l-glutamate, or l-glutamine, respectively, and then incubated at 37° C for 2 h, washed twice with cold SPK buffer, and resuspended in 200 µl SPK buffer (62 (link)). ScintiVerse E cocktail (1 ml; Fisher Scientific, Waltham, MA) was added to each sample. Data were acquired with a model 1450 liquid scintillation counting (LSC) luminescence counter (PerkinElmer).

Cheng Z., Lin M, & Rikihisa Y. (2014). Ehrlichia chaffeensis Proliferation Begins with NtrY/NtrX and PutA/GlnA Upregulation and CtrA Degradation Induced by Proline and Glutamine Uptake. mBio, 5(6), e02141-14.

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Glutamate Uptake in iPSC-Derived Astrocytes

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Glutamate uptake in iPS astrocytes was measured using 0.5 µM L-glutamate (cold:radioactive=99:1) and 0.3 µCi L-[³H]glutamate per sample (PerkinElmer). Cells grown in 6 well tissue culture plates were washed and pre-incubated at RT for 10 min in Na⁺ buffer (5 mM Tri-HCl, pH 7.2, 10 mM HEPES, 140 mM NaCl, 2.5 mM KCl, 1.2 mM CaCl₂, 1.2mM MgCl₂, 1.2 mM K₂HPO₄, and 10 mM D-glucose). Glutamate uptake was measured for 5 min at 37 C in Na⁺ buffer followed by two washes with ice-cold Na⁺ -free assay buffer (5 mM TrisHCl, pH 7.2, 10 mM HEPES, 140 mM Choline-Cl, 2.5 mM KCl, 1.2 mM CaCl₂, 1.2 mM MgCl₂, 1.2 mM K₂HPO₄, and 10 mM D-glucose). To measure the amount of glutamate taken up, cells were lysed with 0.1N NaOH and [³H] was measured using a liquid scintillation counter. Radioactive counts were normalized to total protein levels measured using the Bradford method.

Zhang P.W., Haidet-Phillips A.M., Pham J.T., Lee Y., Huo Y., Tienari P.J., Maragakis N.J., Sattler R, & Rothstein J.D. (2015). Generation of GFAP::GFP astrocyte reporter lines from human adult fibroblast-derived iPS cells using zinc-finger nuclease technology. Glia, 64(1), 63-75.

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Glutamate Uptake Assay in Cultured Astrocytes

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Cultured astrocytes were washed, followed by a pre-incubation at RT for 10 min in Na+ buffer (5mM Tris-HCl, pH 7.2, 10mM HEPES, 140mM NaCl, 2.5mM KCl, 1.2mM CaCl2, 1.2mM MgCl2, 1.2mM K2HPO4, and 10mM D-glucose). Glutamate uptake measurements were initiated by incubating the cells for 5 minutes at 37°C in Na+ buffer containing 0.5 μM L-glutamate and 0.3 μCi L-[3H]glutamate (PerkinElmer) per sample (glutamate ratio cold:radioactive=99:1). The cells were quickly moved onto ice and rapidly washed twice with ice-cold glutamate-free Na+-free assay buffer (5mM Tris-HCl, pH 7.2, 10mM HEPES, 140mM Choline-Cl, 2.5mM KCl, 1.2mM CaCl2, 1.2mM MgCl2, 1.2mM K2HPO4, and 10mM D-glucose). Cells were lysed with 0.1N NaOH solution and [3H] radioactivity was measured using a scintillation counter. Radioactive counts were normalized to total protein levels as determined by Bradford method.

Abazyan S., Yang E.J., Abazyan B., Xia M., Yang C., Rojas C., Slusher B., Sattler R, & Pletnikov M. (2014). Mutant Disrupted-In-Schizophrenia 1 in astrocytes: focus on glutamate metabolism. Journal of neuroscience research, 92(12), 1659-1668.

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Mouse Hippocampal Glutamate Uptake Assay

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Mouse hippocampal tissues were homogenized in ice-cold 0.32M sucrose solution. The tissue lysate was first centrifuged at 800 x g for 10min, followed by a centrifugation at 20,000 x g for 20min at 4°C. Cell pellets were washed in 0.32M sucrose, re-centrifuged and re-suspended in ice-cold 0.32M sucrose. Glutamate uptake was initiated by adding synaptosomes in Na+ buffer (5mM Tris-HCl, pH 7.2, 10mM HEPES, 140mM NaCl, 2.5mM KCl, 1.2mM CaCl2, 1.2mM MgCl2, 1.2mM K2HPO4, and 10mM D-glucose) containing 0.5 μM L-glutamate and 0.075 μCi L-[3H]glutamate (PerkinElmer; glutamate ratio cold:radioactive=99:1) per sample and incubated for 3 minutes at 37°C. The uptake was stopped by quickly adding four volumes of ice-cold glutamate-free Na+-free buffer (5mM Tris-HCl, pH 7.2, 10mM HEPES, 140mM Choline-Cl, 2.5mM KCl, 1.2mM CaCl2, 1.2mM MgCl2, 1.2mM K2HPO4, and 10mM D-glucose). The synaptosome lysate was filtered through a Brandel Harvester and washed with Na+-free assay buffer. The filter paper (containing the labeled synaptosomes) was transferred into scintillation vials and radioactivity was measured using a scintillation counter. Radioactive counts were normalized to total protein levels as determined by Bradford method.

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Evaluating Metabolic Responses in Astrocytes

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Glucose uptake was measured in unstimulated and stimulated astrocytes (50 ng/ml TNFα and 10 ng/ml IL-1β; 48 h) via incorporation of the fluorescent glucose analog 2-NBDG (Thermo Fisher Scientific). 5 µM 2-NBDG was added to cells in low glucose medium (1 g/L D-Glucose) for 30 min. After washing with HBSS, intracellular fluorescence was determined with an Infinite M1000 fluorescent plate reader (Tecan). Lactate secretion was determined from stimulated and unstimulated cell culture supernatants using an enzymatic assay, which produces a colorimetric (570 nm)/fluorometric (λ_ex = 35 nm/λ_em = 587 nm) product proportional to lactate content. (Sigma Aldrich). To quantify glutamate uptake, we incubated astrocyte cultures with HBSS buffer containing 0.5 μM L-glutamate and L-[³H] glutamate (1 μCi; PerkinElmer) at a 100:1 ratio for 5 min at 37 °C. Cells were rapidly moved onto ice, washed twice with ice-cold glutamate-free HBSS buffer, and lysed with 0.1 N NaOH solution. [³H] radioactivity was measured using a scintillation counter and counts were normalized to total protein levels per sample^{29 (link)}.

Ponath G., Lincoln M.R., Levine-Ritterman M., Park C., Dahlawi S., Mubarak M., Sumida T., Airas L., Zhang S., Isitan C., Nguyen T.D., Raine C.S., Hafler D.A, & Pitt D. (2018). Enhanced astrocyte responses are driven by a genetic risk allele associated with multiple sclerosis. Nature Communications, 9, 5337.

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Astrocyte Glutamate Uptake Assay

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Mouse primary astrocyte cell cultures from control, Epm2a−/− and Epm2b−/− mice were seeded 48h before the assay at a density of 2 × 10⁵ cells/well in a 48 wells plate and grown in Dulbecco’s modified Eagle’s medium (Lonza, Barcelona, Spain), supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mM glutamine and 10% of inactivated fetal bovine serum (Lonza, Barcelona, Spain) in a humidified atmosphere at 37°C with 5% CO₂. Cells were washed twice with PBS at 37°C and then incubated with L-glutamate [2 μCi/ml L− [³H]glutamate (PerkinElmer, Madrid Spain)] in PBS at 37°C for 10 min. Incubations were carried out in triplicate. For each assay, parallel determinations were performed in a sodium-free PBS (NaCl was iso-osmotically substituted by choline chloride). Assays were stopped by aspiration followed by two washes with ice-cold PBS or sodium-free PBS respectively. Cells were lysed in 0.2 M NaOH and accumulated radioactivity measured in a liquid scintillation counter (PerkinElmer Liquid Scintillation Analyzer Tri-Carb 2800TR). Glutamate uptake was expressed as the fold change in radioactivity incorporation between cells treated with PBS and cells treated with sodium-free PBS, as sodium is required for the GLT-1 mediated co-transport of glutamate. Each experiment was repeated at least three times.

Muñoz-Ballester C., Berthier A., Viana R, & Sanz P. (2016). Homeostasis of the astrocytic glutamate transporter GLT-1 is altered in mouse models of Lafora disease. Biochimica et biophysica acta, 1862(6), 1074-1083.

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