Synaptic vesicles were purified as previously described14 (link). The standard buffer for glutamate uptake contained 148 mM choline or potassium gluconate, 2 mM choline or KCl, 4 mM MgATP, 10 mM HEPES-Tris, pH 7.4, 1 mM choline glutamate, and 40 μCi/ml [3H]l -glutamate (Perkin-Elmer). The standard buffer for γ-aminobutyric acid (GABA) uptake contained 145 mM choline or potassium gluconate, 5 mM KCl, 4 mM MgATP, 10 mM HEPES-Tris, pH 7.4, 50 µM GABA, and 65 μCi/ml [3H]GABA (Perkin-Elmer). Transport was initiated by adding 100 μg LP2 protein to 200 μl reaction buffer (pre-warmed to 30 °C). The reaction was incubated at 30 °C for 10 min and stopped by rapid filtration and washing four times with 2 ml cold reaction buffer lacking glutamate/GABA. Bound radioactivity was detected by liquid scintillation, and background transport measured in the presence of 100 μM Evans Blue (glutamate) or 5 μM nigericin + 20 μM valinomycin (GABA) was subtracted. In every experiment, each condition was assayed in triplicate and at least three independent experiments were performed using at least three different synaptic vesicle preparations. Results are presented as mean ± s.e.m and the statistics analyzed by two-way ANOVA test with Bonferroni post hoc comparison.
3h gaba
Manufactured by PerkinElmer
Sourced in United States, United Kingdom
[3H]GABA is a radioactively labeled gamma-aminobutyric acid (GABA) compound used in research applications. It serves as a tool for studying GABA-related processes and mechanisms in various biological systems.
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Lab products found in correlation
3h dopamine, by PerkinElmer (2 mentions)
3h serotonin, by PerkinElmer (2 mentions)
3h norepinephrine, by PerkinElmer (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
L 3h glutamate, by PerkinElmer (1 mentions)
Aminooxyacetic acid, by Merck Group (1 mentions)
Baclofen, by Bio-Techne (1 mentions)
Cgp 52432, by Bio-Techne (1 mentions)
Gf109203x, by Merck Group (1 mentions)
Alexa fluor 488 goat anti guinea pig, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 555 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Kapa mouse genotyping kit, by Roche (1 mentions)
Ly367385, by Bio-Techne (1 mentions)
Rs 3 5 dhpg, by Bio-Techne (1 mentions)
2 3 3h d aspartate, by PerkinElmer (1 mentions)
Donkey antimouse alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
14c choline, by PerkinElmer (1 mentions)
Cell harvester, by Brandel Inc (1 mentions)
9 protocols using 3h gaba
1
Synaptic Vesicle Glutamate and GABA Uptake
2
Radioligand Binding and Western Blotting
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[2,3-3H]D -aspartate (specific activity 11.3 Ci/mmol) and [3H]GABA (specific activity 30.0 Ci/mmol) were from Perkin Elmer (Boston, MA, United States). (±)-baclofen, LY367385, (RS)-3,5 DHPG, and CGP 52432 were purchased from Tocris Bioscience (Bristol, United Kingdom). GF109203X, aminooxyacetic acid, naphtol blue black staining, horseradish peroxidase-coupled anti-mouse and anti-rabbit secondary antibodies were from Sigma (Milan, Italy). Donkey anti-mouse AlexaFluor-647, goat anti-guinea pig AlexaFluor-488, goat anti-rabbit AlexaFluor-555 were from Life Technologies Corporation (Carlsbad, CA, United States). Mouse anti-GABAB1 and mouse anti-GABAB2 antibodies were from Santa Cruz Biotechnology (Dallas, TX, United States). Rabbit anti-mGlu1 antibody, used in confocal analysis, was from Abcam (Cambridge, United Kingdom) and mouse anti-mGlu1 monoclonal antibody, used in Western blotting, was from BD Biosciences (San Jose, CA, United States). Guinea pig anti-vesicular glutamate transporters type 1 antibody was from Millipore (Temecula, CA, United States). Guinea pig anti-VGAT was from AlomoneLabs (Jerusalem, Israel). Bradford assay was from Bio-Rad (Segrate, Milan, Italy). KAPA Mouse Genotyping Kits were from Kapa Biosystems (Woburn, MA, United States). ECL AdvanceTM was from Amersham Biosciences (Piscataway, NJ, United States).
3
Quantifying neurotransmitter release in irradiated rats
4
GABA Binding Assay in Xenopus Oocytes
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Membrane proteins from Xenopus oocyte were prepared and [3H]-GABA binding was performed as described previously58 (link). Oocytes were homogenized in 2 ml of extraction buffer [pH 7.2, 0.32 mM sucrose, 100 μM EDTA, 1% proteinase inhibitor mixture I (Sigma)]. The homogenate was centrifuged at 1,000 × g for 30 min. The resultant supernatant was filtered through four layers of cheesecloth and centrifuged at 30,000 × g for 60 min. The pellet was resuspended in the incubation buffer (pH 7.4, 0.05 mM Tris, 0.12 mM NaCl, 100 μM EDTA). [3H]-GABA (33.4 Ci/mmol) was purchased from Perkin-Elmer Life and Analytical Sciences (Waltham, MA). Samples were assayed by filtration onto Whatman GF/B filters presoaked in 0.5% polyethylenimine, followed by rapid washing by using a cell harvester (Brandel, Bethesda, MD). Amounts of total protein were determined by a Bio-Rad DC protein assay using BSA standards.
5
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).
6
GABA Binding Assay Reagents
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[3H]GABA (specific activity: 25–40 Ci/mmol) was obtained from PerkinElmer Life Sciences (Llantrisant, United Kingdom). Liothyronine was purchased from Tocris Bioscience (Bristol, United Kingdom). 4-Phenylbutyrate (4-PBA), liothyronine and tiagabine were obtained from Sigma-Aldrich (St. Louis, MO, USA). Cell culture media, supplements, and antibiotics were obtained from Invitrogen. SDS was from BioMol GmbH (Hamburg, Germany). Bovine serum albumin (BSA) and Complete TM protease inhibitor mixture were purchased from Roche Applied Science (Mannheim, Germany). Tris and scintillation mixture (Rotiszint® eco plus) were ordered from Carl Roth GmbH (Karlsruhe, Germany). Anti-green fluorescent protein (GFP) antibodies (ab290 and A-11122) were purchased from Abcam Plc (Cambridge, UK) and Invitrogen (Life Technologies, Carlsbad, CA, United States), respectively. All other chemicals were of analytical grade. The MNCD2: hybridoma, monoclonal antibody (developed by M. Takeichi and H. Matsunami) was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology (Iowa City, IA 52242, USA).
7
Radiolabeled Neurotransmitter Uptake Assay
8
Measuring GABA Release in Mouse ARC
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We euthanized ad libitum–fed and 48 h–fasted mice by live decapitation. We extracted brains, placed them briefly in cold PBS, and sectioned them into 1-mm coronal slices by using a stainless steel mouse brain matrix. We excised small punches of tissue corresponding to the known location of the ARC, as compared with the mouse brain atlas (Paxinos and Franklin, 2001 ), using a 15-gauge needle. We incubated the ARC microdissections on Krebs-Ringer bicarbonate buffer (KRBB) saturated with 95% O2 and 5% CO2 for 10 min at 37°C. Next, we incubated with [3H]GABA (367 cpm/ml, with a specific activity of 92.1 Ci/mmol; PerkinElmer) in KRBB for 20 min to fill synaptic vesicles with the tracer, and we performed two washes with KRBB. After that, we incubated ARC microdissections for 10 min with fresh KRBB containing 56 mM KCl. Finally, we collected the medium, mixed it with 150 µl scintillator (Ecolite), and measured the radioactivity in a β-counter (Tracor Analytic).
9
GABA Release Assay in iCell GABANeurons
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iCell GABANeurons (Cellular Dynamics International, USA) were plated at a density of 60,000 cells per well in 96-well plates coated with poly-l -ornithine and laminin (Sigma-Aldrich, UK). iCell GABANeurons were maintained according to the manufacturer’s instructions. iCell GABANeurons were fed every 3 to 4 days by replacement of half media.
GABA release was assessed in iCell GABANeurons at DIV 16 to 18. Cells were treated with neurotoxins for 24 hours at 37°C. Following removal of neurotoxin, cells were washed three times in Neurobasal medium containing 1% B27 and 0.5 mM GlutaMAX (assay medium). Cells were loaded with [3H]-GABA (2 μCi/ml; PerkinElmer, UK) in assay medium for 120 min at 35°C. Following removal of [3H]-GABA, cells were washed three times with assay medium. Basal and stimulated [3H]-GABA release was established by incubation at 35°C for 5 min with assay medium (50 μl per well) containing low potassium (5.3 mM KCl) or high potassium (60 mM KCl), respectively. To determine retained [3H]-GABA, cells were lysed by adding radioimmunoprecipitation assay buffer (50 μl per well; Sigma-Aldrich, UK). Superfusates and cell lysates were transferred into 96-well IsoPlates (PerkinElmer, UK), and OptiPhase Supermix scintillation fluid (200 μl per well) was added. Radioactivity was quantified using a MicroBeta2 plate reader (PerkinElmer, UK).
GABA release was assessed in iCell GABANeurons at DIV 16 to 18. Cells were treated with neurotoxins for 24 hours at 37°C. Following removal of neurotoxin, cells were washed three times in Neurobasal medium containing 1% B27 and 0.5 mM GlutaMAX (assay medium). Cells were loaded with [3H]-GABA (2 μCi/ml; PerkinElmer, UK) in assay medium for 120 min at 35°C. Following removal of [3H]-GABA, cells were washed three times with assay medium. Basal and stimulated [3H]-GABA release was established by incubation at 35°C for 5 min with assay medium (50 μl per well) containing low potassium (5.3 mM KCl) or high potassium (60 mM KCl), respectively. To determine retained [3H]-GABA, cells were lysed by adding radioimmunoprecipitation assay buffer (50 μl per well; Sigma-Aldrich, UK). Superfusates and cell lysates were transferred into 96-well IsoPlates (PerkinElmer, UK), and OptiPhase Supermix scintillation fluid (200 μl per well) was added. Radioactivity was quantified using a MicroBeta2 plate reader (PerkinElmer, UK).
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