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3h glutamate

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3H-glutamate is a radiolabeled amino acid used in research applications. It serves as a tracer for the study of glutamate metabolism and neurotransmission.

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Lab products found in correlation

Liquid scintillation counter, by Beckman Coulter (2 mentions) Bradford protein assay, by Bio-Rad (1 mentions) 3h histamine, by PerkinElmer (1 mentions) 3h gaba, by PerkinElmer (1 mentions) 3h dopamine, by PerkinElmer (1 mentions) 3h serotonin, by PerkinElmer (1 mentions) 3h acetate, by American Radiolabeled Chemicals (1 mentions) 3h norepinephrine, by PerkinElmer (1 mentions) 3h choline chloride, by PerkinElmer (1 mentions) 3h atp, by PerkinElmer (1 mentions) Collagenase d, by Serva Electrophoresis (1 mentions) 3h dhea, by PerkinElmer (1 mentions) 3h taurocholic acid, by American Radiolabeled Chemicals (1 mentions) C2978, by Merck Group (1 mentions) Ls6500 scintillation counter, by Beckman Coulter (1 mentions) Bradford protein assay kit, by Bio-Rad (1 mentions)

7 protocols using 3h glutamate

1

Glutamate Uptake Measurement in Brain Slices

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Glutamate uptake was measured using an in vitro slice preparation as described previously26 (link). Briefly, two weeks after stress or sham animals received daily ceftriaxone (200 mg/kg, ip) or vehicle (saline) for 5 days prior to bilateral dissection of the NAcore or dorsolateral striatum. The tissue was sliced into 250 × 250 µm sections, and each hemisphere incubated at 37°C in either oxygenated Kreb's-Ringer's solution phosphate buffer (KRP) (in mM: 140 NaCl, 1.2 CaCl2, KH2PO4, 5 HEPES, 10 glucose, and 1 MgCl2) for Na+-dependent glutamate uptake, or in KRP buffer with NaCl replaced by 140 mM choline chloride for Na+-independent glutamate uptake. Glutamate uptake measurements were initiated by adding 3H-glutamate (40 nM, 50 Ci/mmol; PerkinElmer) in the presence of unlabeled glutamate (10 µM) in a final volume of 250 µl of KRP buffer. The reaction was conducted at 37°C for 15 min, and was terminated by washing the slices with ice-cold sodium-free buffer. The tissue was then solubilized using 1% SDS, and radioactivity and protein content quantified. Counts per minute were converted to uptake/mg protein/15 min.
Garcia-Keller C., Kupchik Y., Gipson C.D., Brown R.M., Spencer S., Bollati F., Esparza M.A., Roberts-Wolfe D., Heinsbroek J., Bobadilla A.C., Cancela L.M, & Kalivas P.W. (2015). Glutamatergic Mechanisms of Comorbidity Between Acute Stress and Cocaine Self-administration. Molecular psychiatry, 21(8), 1063-1069.
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2

Measurement of System x(c)- Activity

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For measurement of system xc − activity, 2.5 × 104 HT22 were plated in 24-well dishes. After 24 h, the cells were washed three times with sodium-free Hank’s balanced salt solution (HBSS). System xc− activity was measured as sodium-insensitive, homocysteic acid-inhibitable uptake of [3H]glutamate (Perkin Elmer, Waltham, MA, USA) as described [44 (link)]. Briefly, cells in triplicate were incubated in 10 μM glutamate ([3H]glutamate/cold glutamate 1:1000) with or without 1 mM homocysteic acid adjusted to pH 7.4 in sodium-free HBSS. Cells were washed three times with ice-cold, sodium-free HBSS and lysed in 0.2 N NaOH. Radioactivity was measured by liquid scintillation counting and normalized to protein measured by the Bradford method (Pierce).
Dafre A.L., Goldberg J., Wang T., Spiegel D.A, & Maher P. (2015). Methylglyoxal, the foe and friend of glyoxalase and Trx/TrxR systems in HT22 nerve cells. Free radical biology & medicine, 89, 8-19.
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3

Glutamate Uptake Assay in iPSA Cells

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After transduction with lentiviral-GFP vector or lentiviral-GLT1 vector, hIPSAs were cultured for 10 days. Human fibroblasts transduced with lentiviral-GFP vector were used as control. Glutamate uptake activity was measured as previously described (Dowd and Robinson, 1996 (link)), with slight modification. Briefly, cells were washed and pre-incubated with either a sodium- or choline-containing uptake buffer (in mM: Tris, 5; HEPES, 10; NaCl or choline chloride, 140; KCl, 2.5; CaCl2, 1.2; MgCl2, 1.2; K2HPO4, 1.2; glucose, 10) for 20 min at 37°C; and in DHK treatment groups, 100µM of DHK was added to inhibit GLT1. The uptake buffer was then replaced with fresh uptake buffer containing 20nM 3H-glutamate (49 Ci/mmol; PerkinElmer, CA) and 20µM unlabeled glutamate. The cells were incubated for 5 minutes at 37°C. The reaction was terminated by washing cells three times with choline-containing uptake buffer containing 2mM unlabeled glutamate, followed by immediate lysis in ice-cold 0.1N NaOH. Cell extracts were then measured with a liquid scintillation counter (Beckman Instruments, Fullerton, CA). The protein content in each well was measured using the Bradford protein assay (Bio-Rad, Hercules, CA).
Li K., Javed E., Scura D., Hala T.J., Seetharam S., Falnikar A., Richard J.P., Chorath A., Maragakis N.J., Wright M.C, & Lepore A.C. (2015). Human iPS cell-derived astrocyte transplants preserve respiratory function after spinal cord injury. Experimental neurology, 271, 479-492.
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4

Radiolabeled Compounds for Neurotransmitter Assays

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All of the chemicals, unless otherwise stated, were from Sigma-Aldrich (Taufkirchen, Germany). Citalopram was purchased from Biotrend (Cologne, Germany) and collagenase D was from Serva (Heidelberg, Germany). [3H]DHEAS (70.5 Ci/mmol), [3H]E-3-S (45.6 Ci/mmol), [3H]aspartate (11.3 Ci/mmol), [3H]GABA (76.2 Ci/mmol), [3H]histamine (13.4 Ci/mmol), [3H]choline chloride (66.7 Ci/mmol), [3H]norepinephrine (56.6 Ci/mmol), [3H]serotonin (28.25 Ci/mmol), [3H]dopamine (38.7 Ci/mmol), [3H]glutamate (49.6 Ci/mmol), [3H]ATP (30.9 Ci/mmol), [35S]Adenosine 5′-(γ-thio) triphosphate (12.5 mCi/mmol) and [3H]acetylcholine iodide (99.7 Ci/mmol) were purchased from PerkinElmer Life Sciences (Boston, MA, USA). [3H]PREGS (20 Ci/mmol), [3H]taurocholic acid (10.0 Ci/mmol), [3H]acetate (150 mCi/mmol), and [3H]lithocholic acid (50 Ci/mmol) were obtained from American Radiolabeled Chemicals (St. Louis, MO, USA).
Schmidt S., Moncada M., Burger S, & Geyer J. (2015). Expression, sorting and transport studies for the orphan carrier SLC10A4 in neuronal and non-neuronal cell lines and in Xenopus laevis oocytes. BMC Neuroscience, 16, 35.
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5

Glutamate Uptake Assay in Cultured Astrocytes

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Cultured astrocytes in 24-well plates were washed twice and then incubated in Krebs buffer (135 mM NaCl, 5 mM KCl, 0.6 mM MgSO4, 1 mM CaCl2, 6 mM d-glucose, and 10 mM HEPES; pH 7.5) containing 20 nM 3H-glutamate (0.5 μCi; specific activity = 50.8 Ci/mmol; PerkinElmer) and 40 μM unlabeled glutamate for 10 minutes at 37°C. DHK was used to distinguish DHK-sensitive (EAAT2) 3H-glutamate uptake from DHK-insensitive uptake. After incubation, the Krebs buffer was replaced with PBS to stop the uptake of glutamate, and the cells were then collected in cell lysis solution (C2978; Sigma-Aldrich). The lysates were processed to measure both 3H-glutamate levels with a liquid scintillation counter (LS6500 scintillation counter; Beckman Coulter) and protein content by using the Bradford Protein Assay kit (5000002; Bio-Rad). The concentration of glutamate taken up by the astrocytes (in nanomolars) was calculated using the radioactivity and the total protein concentration per milligram of protein over a 10-minute period. DHK-sensitive 3H-glutamate uptake was calculated as the total amount of Na+ dependent 3H-glutamate taken up minus the amount of DHK-insensitive Na+-dependent 3H-glutamate taken up (DHK-treated samples). All values were normalized to mean glutamate uptake in the control group, and all measurements were repeated 3 times.
Sha L., Li G., Zhang X., Lin Y., Qiu Y., Deng Y., Zhu W, & Xu Q. (2022). Pharmacological induction of AMFR increases functional EAAT2 oligomer levels and reduces epileptic seizures in mice. JCI Insight, 7(15), e160247.
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6

Glutamate Uptake Measurement in Brain Slices

Cited 1 time
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Glutamate uptake was measured using an in vitro slice preparation as described previously26 (link). Briefly, two weeks after stress or sham animals received daily ceftriaxone (200 mg/kg, ip) or vehicle (saline) for 5 days prior to bilateral dissection of the NAcore or dorsolateral striatum. The tissue was sliced into 250 × 250 µm sections, and each hemisphere incubated at 37°C in either oxygenated Kreb's-Ringer's solution phosphate buffer (KRP) (in mM: 140 NaCl, 1.2 CaCl2, KH2PO4, 5 HEPES, 10 glucose, and 1 MgCl2) for Na+-dependent glutamate uptake, or in KRP buffer with NaCl replaced by 140 mM choline chloride for Na+-independent glutamate uptake. Glutamate uptake measurements were initiated by adding 3H-glutamate (40 nM, 50 Ci/mmol; PerkinElmer) in the presence of unlabeled glutamate (10 µM) in a final volume of 250 µl of KRP buffer. The reaction was conducted at 37°C for 15 min, and was terminated by washing the slices with ice-cold sodium-free buffer. The tissue was then solubilized using 1% SDS, and radioactivity and protein content quantified. Counts per minute were converted to uptake/mg protein/15 min.
Garcia-Keller C., Kupchik Y., Gipson C.D., Brown R.M., Spencer S., Bollati F., Esparza M.A., Roberts-Wolfe D., Heinsbroek J., Bobadilla A.C., Cancela L.M, & Kalivas P.W. (2015). Glutamatergic Mechanisms of Comorbidity Between Acute Stress and Cocaine Self-administration. Molecular psychiatry, 21(8), 1063-1069.
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7

Radiolabeled Glutamate Uptake Assay

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Radiolabeled glutamate uptake assays were performed as described previously (Ashpole et al., 2013 (link)). Following treatment, cells were washed with ice-cold phosphate buffered saline and subsequently stimulated with growth media containing 1 μg/mL [3H]-glutamate (Perkin Elmer) and 100 μM unlabeled glutamate for varying lengths of time at 37°C. The treatments (with drug and with glutamate) were assigned with simple randomization in which all doses or time points had equal n. The cultures were then washed in ice-cold phosphate buffered saline by a researcher blinded to the treatment groups, lysed in 200 mM NaCl, 0.1 mM EGTA, 20 mM Tris HCl pH 7.4, mixed, and diluted in 3 mL RadSafe liquid scintillation fluid (Beckman Coulter). [3H]-Glutamate within the lysates was quantified using a Beckman liquid scintillation counter.
Prabhu D., Khan S.M., Blackburn K., Marshall J.P, & Ashpole N.M. (2019). Loss of insulin-like growth factor-1 signaling in astrocytes disrupts glutamate handling. Journal of neurochemistry, 151(6), 689-702.
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