Mice were anesthetized using isofluorane and then decapitated. Brains were removed, cut into left and right hemispheres, and then 300 μm coronal slices were made at 1-4°C in oxygenated (95% v/v O2, 5% v/v CO2) dissecting solution (208 mM sucrose, 2.5 mM KCl, 1 mM CaCl2, 4 mM MgCl2, 4 mM MgSO4, 1.6 mM NaH2PO4, 26 mM NaHCO3, 10 mM glucose, and 3 mM Na-pyruvate) using a Vibratome 3000 (The Vibratome Company). Typically, a total of 5-7 striatal hemi-slices (left/right hemisphere combined) were obtained from each mouse brain (1.1 to 0.14 mm from Bregma). Slices were allowed to recover on a nylon mesh for 1 h at 30°C in oxygenated ACSF (113 mM NaCl, 2.5-5 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1 mM NaH2PO4, 26 mM NaHCO3, 20 mM glucose, and 3 mM Na-pyruvate) followed by addition of picrotoxin (50 μM) for 30 min. Slices were then transferred to oxygenated 30°C ACSF solutions supplemented with vehicle or drug for 1-30 min, as defined in the figure legends. The following drugs were used and dissolved in water or DMSO based on manufacturer's instructions: BAPTA (Sigma), (S)-BayK8644 (Tocris), FPL64176 (Tocris), isradipine (National Institute of Mental Health Chemical Synthesis and Drug Supply Program), KCl (Sigma), mibefradil (Tocris), Nickel Chloride (Sigma), nimodipine (MP Biomedicals), SNX-482 (Peptides International), and TTX (Tocris).
Fpl64176
Manufactured by Bio-Techne
Sourced in United Kingdom, United States
The FPL64176 is a piece of lab equipment manufactured by Bio-Techne. It is designed for use in laboratory settings, but a detailed and unbiased description of its core function cannot be provided while maintaining conciseness.
Automatically generated - may contain errors
Lab products found in correlation
Gr73632, by Bio-Techne (5 mentions)
Isradipine, by Bio-Techne (2 mentions)
Thapsigargin, by Santa Cruz Biotechnology (2 mentions)
Dantrolene, by Santa Cruz Biotechnology (2 mentions)
2 apb, by Santa Cruz Biotechnology (2 mentions)
Gf109203x, by Bio-Techne (2 mentions)
Thapsigargin, by Bio-Techne (2 mentions)
Sr141716a, by Bio-Techne (2 mentions)
Quinpirole hcl, by Merck Group (2 mentions)
Mcn a 343, by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Nickel chloride, by Merck Group (1 mentions)
Mibefradil, by Bio-Techne (1 mentions)
Bapta, by Merck Group (1 mentions)
S bayk8644, by Bio-Techne (1 mentions)
Snx 482, by Peptide Institute (1 mentions)
Potassium chloride (kcl), by Merck Group (1 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (1 mentions)
Riluzole, by Bio-Techne (1 mentions)
ω agatoxin iva, by Alomone (1 mentions)
10 protocols using fpl64176
1
Striatal Slice Preparation and Neuromodulation
2
Stimulation of Cortical Neurons
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Prior to stimulation, neurons were first placed overnight into a minimal defined medium (Papadia et al., 2005 (link)) containing 10% MEM (Invitrogen), 90% Salt-Glucose-Glycine (SGG) medium ([Bading et al., 1993 (link)]; SGG: 114 mM NaCl, 0.219% NaHCO3, 5.292 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM HEPES, 1 mM Glycine, 30 mM Glucose, 0.5 mM sodium pyruvate, 0.1% Phenol Red; osmolarity 325 mosm/l,[Papadia et al., 2005 (link)]). KCl/FPL stimulations were performed as described previously (Hardingham et al., 1999 (link), 1997 (link)). KCl/FPL stimulation involved neurons being exposed to 50 mM KCl by adding 0.41 volumes of KCl depolarization solution (Bading et al., 1993 (link)) (10 mM HEPES, pH 7.2, 170 mM KCl, 1 mM MgCl2, 2 mM CaCl2) in the presence of 5 µM FPL64176 (Tocris), plus an NMDA receptor antagonist (MK-801, 5 µM) to prevent any excitotoxicity. As described recently (Bilican et al., 2014 (link)), hESCCORT-neurons respond strongly and uniformly to KCl/FPL treatment, with over 80% of hESCCORT-neurons exhibiting a >10-fold increase in [Ca2+], and the remainder showing at least a 5-fold increase.
3
Electrophysiological Assessment of Neurotransmitter Modulation
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MEA experiments were performed at 37°C in standard cell culture medium or aCSF. Gabazine, D-AP-5, and NBQX (end concentration: 50 μM each) were consecutively added to this extracellular solution. Riluzole (1, 2, 5, or 40 μM), isradipine (4 μM), or FPL 64176 (2 μM; all from Tocris Bioscience) were applied after the inhibition of excitatory and inhibitory FSC. Dose responses were analyzed by consecutively increasing the concentration of AP-5, NBQX, or Riluzole with 2 min of MEA recording and 5 min incubation time after application.
4
Botulinum Neurotoxin Activation and Reagents
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BoNT/A1 (2.5 × 108 MLD50/mg), BoNT/D1 (2.8 × 107 MLD50/mg) and BoNT/E1 (3.0 × 107 MLD50/mg) were purchased from Metabiologics (Madison, WI, USA). For BoNT/E, toxin was activated by a 60 min incubation at 37 °C with 0.3 mg/mL TPCK-treated trypsin in 0.05 M sodium phosphate buffer (pH 6.5)19 (link). ω-agatoxin IVA, ω-conotoxin MVIIC and GV-58 were purchased from Alomone Labs (Jerusalem, Israel). FPL 64176 was purchased from Tocris Bioscience (Bristol, UK). All reagents for electrophysiology buffers were obtained from Sigma-Aldrich (St. Louis, MO, USA).
5
Pharmacological Modulators of Cellular Signaling
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The following drugs were used: m-3M3FBS, U73122, U0126, GF109203X, FPL64176, 2-methyl-serotonin maleate salt (2-Methyl-5-HT), and GR73632 were purchased from Tocris (Minneapolis, MN); McN-A-343, quinpirole HCl and nifedipine from Sigma Sigma/RBI (St. Louis, MO); thapsigargin, dantrolene and 2-APB from Santa Cruz Biotechnology (Dallas, TX). Palonosetron and netupitant were kindly provided by Helsinn Health Care (Lugano, Switzerland). m-3M3FBS, U73122, nifedipine, U0126, netupitant were dissolved in a mixture of ethanol/Tween 80/saline at a volume ratio of 1:1:18. dantrolene, 2-APB, GF109203X and FPL64176 were dissolved in 25% DMSO in water. thapsigargin was dissolved in 10% DMSO in distilled water. Other drugs were dissolved in distilled water. All drugs were administered at a volume of 0.1 ml/10 g of body weight.
6
Pharmacological Inhibition of Cell Signaling
7
Pharmacological Characterization of Emetogens
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Temsirolimus, everolimus, apomorphine HCl, GR73632, SR141716A, thapsigargin, and FPL64176 were purchased from Tocris (Minneapolis, MN). Ridaforolimus and rapamycin were acquired from MedChemExpress and Calbiochem, respectively. Quinpirole HCl, serotonin HCl (5-HT), 2-methyl-serotonin maleate salt (2-methyl-5-HT), pilocarpine HCL, McN-A-343, and cisplatin (cis-platinum (II) diamine dichloride (Pt (NH3)2)Cl2) were obtained from Sigma/RBI. Apomorphine, quinpirole, serotonin, 2-methy-5-HT, McN-A-343, GR73632, pilocarpine, and cisplatin were dissolved in distilled water. thapsigargin was dissolved in 10% DMSO (Sigma) in water. mTOR inhibitors and FPL64176 were dissolved in DMSO and then diluted with three volumes of distilled water to a final DMSO concentration of 25%. SR141716A was dissolved in a 1:1:18 solution of ethanol, emulphor (EL-620, a polyoxyethylated vegetable oil, GAF Corporation, Linden, NJ), and 0.9% saline. All drugs were administered at a volume of 0.1 ml/10 g of body weight. The doses and routes used for the emetogens were based upon previous publications from our laboratory (Darmani et al., 2019 (link); Darmani et al., 2020 (link); Zhong and Darmani 2020 (link)).
8
Emetic Dose Response Evaluation
9
Pharmacological Agents for Emetic Study
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The following drugs were used in the present study: clonidine, dexmedetomidine, yohimbine, GR73632, SR141716A, FPL64176, thapsigargin, and ZD7288 were purchased from Tocris (Minneapolis, MN, USA); McN-A-343, quinpirole HCl, and rolipram were purchased from Sigma-Aldrich (St. Louis, MO, USA); 2-methyl-serotonin maleate salt (2-Methyl-5-HT) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). SR141716A was dissolved to twice the stated drug dose in a 1:1:18 solution of ethanol:Emulphor™:0.9% saline and was then diluted further with an equal volume of saline. FPL64176 was dissolved in 25% DMSO in water. thapsigargin was dissolved in 10% DMSO in distilled water. Other drugs were dissolved in distilled water. All drugs were administered at a volume of 0.1 mL/10 g of body weight. The doses and routes used for the emetogens were based upon previous publications from our laboratory [6 (link),43 (link),87 (link)].
10
Pharmacological Agents in Experimental Studies
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