For volatile anesthetic treatment, coverslips in 12-well plates with a low volume of culture media (500μl to facilitate gas diffusion, were placed in airtight, humidified modular chambers (Billups-Rothenberg, Del Mar, CA, USA) as previously described (Mintz, et al. 2013 (link); Xu, et al. 2018b (link)). The chamber was connected to an agent-specific calibrated vaporizer (SuperaVet, Vaporizer Sales and Services Inc, Rockmart, GA, USA) that delivered 2.4% isoflurane mixed with 5% carbon dioxide / 95% air carrier gas at 12 L/min. Carrier gas alone was used as for controls. Gas composition was measured periodically using a 5250 RGM gas analyzer (Datex-Ohmeda, Madison, WI, USA). In some cases, co-treatment or pre-treatment with pharmacologic compounds was performed. These compounds included: TCS 1205 (10 nM, 100 nM, 1μM, Tocris), TCS 1105 (10 nM, 100 nM, 1μM, Tocris), Zolpidem (10 nM, 100 nM, 1μM, Tocris), Bumetanide (10μM, Tocris), and Chloride Ionophore I (3μM, Millipore). After a 15-min equilibration, the sealed chambers with dissociated cultures were placed in an incubator to maintain temperature at 37°C for 1 hour, followed by 20 min exposure to either vehicle control or a recombinant soluble axon guidance cue, Semaphorin 3A (R & D Systems, Bend, OR) at 100 ng/ml to induce growth cone collapse (Figure 1A , B ).
Bumetanide
Manufactured by Bio-Techne
Sourced in United Kingdom
Bumetanide is a biochemical compound used in research applications. It functions as a potent loop diuretic that inhibits the sodium-potassium-chloride cotransporter, which is responsible for the reabsorption of these ions in the thick ascending limb of the loop of Henle in the kidney.
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Lab products found in correlation
Furosemide, by Bio-Techne (4 mentions)
Picrotoxin, by Merck Group (4 mentions)
Picrotoxin, by Bio-Techne (3 mentions)
Gabazine, by Bio-Techne (2 mentions)
Vu0240551, by Bio-Techne (2 mentions)
Muscimol, by Bio-Techne (2 mentions)
Benidipine, by Bio-Techne (2 mentions)
Tpmpa, by Bio-Techne (2 mentions)
Vu0463271, by Bio-Techne (2 mentions)
Bumetanide, by Merck Group (2 mentions)
Tcs 1105, by Bio-Techne (1 mentions)
5250 rgm gas analyzer, by GE Healthcare (1 mentions)
Zolpidem, by Bio-Techne (1 mentions)
Semaphorin 3a, by R&D Systems (1 mentions)
4 aminopyridine 4 ap, by Merck Group (1 mentions)
Bicuculline methiodide, by Merck Group (1 mentions)
Tetrodotoxin ttx, by Fujifilm (1 mentions)
Bicuculline methochloride, by Bio-Techne (1 mentions)
Brain derived neurotrophic factor (bdnf), by Santa Cruz Biotechnology (1 mentions)
Trkb fc chimera, by R&D Systems (1 mentions)
23 protocols using bumetanide
1
Volatile Anesthetic Exposure and Growth Cone Collapse
2
Ion Channel Pharmacology in Nerve Recordings
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Control buffer solution contained (in mM): NaCl 140, HEPES hemi Na 10, CaCl2 2.1, MgCl2 2.12, KCl 2.5, Glucose 10. It was adjusted to pH 7.2–7.3 with the addition of small quantities of HCl, as necessary. 4-Ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidinium chloride (ZD7288) and bumetanide were obtained from Tocris (Bio-techne Ltd, Abingdon, Oxfordshire, UK). These drugs were made up as stock solutions in DMSO, at 50 mM and 5 mM, respectively, and stored at − 20 °C. The final concentrations were achieved by one-thousand fold dilution and the concentration of vehicle in the nerve bath never exceeded 0.1%. 4-Aminopyridine (4-AP) was obtained from Sigma-Aldrich (Merck, Poole, Dorset, UK), and was made as a fresh solution from the crystalline form on each experimental day, ensuring appropriate pHing of the buffer solution took place using HCl.
3
Pharmacological Modulation of Neuronal Signaling
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The following drugs were used: JPW1114 (also named di-2 ANEPEQ) (Molecular Probes/Life Technology, Carlsbad, CA, USA); bicuculline methiodide (BIC) and strychnine (STR) (Sigma-Aldrich); 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), DL-2-amino-5-phosphono-pentanoic acid (D-AP5), picrotoxin (PTX), bumetanide (BUM) and 2-(guanidino)ethanesulfonic acid (GES) (Tocris Bioscience, Bristol, UK); tetrodotoxin (TTX) (Wako, Tokyo, Japan).
4
Neurochemical Modulation of TrkB Signaling
5
Electrophysiology Protocols with Tocris Compounds
6
Pharmacological Modulation of GABA Reversal
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Working drug solutions were freshly prepared from stock solutions on the day of the experiments. Kynurenic acid stock solution (500 mM; Sigma-Aldrich) was prepared in 1 N NaOH and stored at 4°C; the drug was used at a final concentration of 3 mM. Picrotoxin stock solution (100 mM; Tocris) was prepared in DMSO and kept at −20°C. The final working solution was 50 μM. Bumetanide (Tocris) stock solution (10 mM in DMSO) was prepared fresh every day. For slice recording of Bumetanide-treated mice, the mice received intraperitoneal injections (0.1 mg/kg in saline) 30 to 40 min before being euthanized, and Bumetanide (10 μM) was also added to the ACSF in the slice incubation and recording chambers. Simply adding Bumetanide to the recording solution did not produce notable effects on the GABAA reversal potential when recording slices from scPCP mice, most likely because Bumetanide requires ≥20 min to significantly affect eGABA (48 (link)), and in our experience, this time span is close to the maximum safe duration of perforated patch recordings.
7
Preparation and Use of Ionic Solutions
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CaCCinh-A01 (Tocris), benzbromarone (Tocris), nicardipine (Sigma), TTA-A2 (Alamone), and isradipine (Tocris) were dissolved in DMSO (concentration of DMSO in solution did not exceed 0.01%). ZD7288 (Tocris) was dissolved in water. Furosemide (Tocris) and bumetanide (Tocris) were dissolved in 100% ethanol (ethanol concentration did not exceed 0.01%). Nominal Ca2+ solutions (0 mM Ca2+ + 1 mM EGTA) contained (in mM): 120.35 NaCl 5.9 KCl, 15.5 NaHCO3, 1.2 Na2HPO4, 1.2 MgCl2, 11.5 glucose, 2.5 MgCl2, and 1.0 EGTA.
8
In Vivo Modulation of NKCC1 in Mice
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Male adult NKCC1fl/fl mice were injected intraperitoneally with saline, LPS (2 mg/kg; O26:B6, #L8274, Sigma-Aldrich) or LPS (2 mg/kg) and bumetanide (25 mg/kg; #3108, Tocris). Intraperitoneal bumetanide injections were repeated twice, the first one 15 minutes prior to LPS injection, the second one 1 hour after LPS administration. The double injection aimed to ensure that in the critical time window, we have effective concentrations of bumetanide in the circulation. At 24 hours, saline-perfused spleen and brain samples were collected for cytokine measurements or flow cytometric analysis.
9
Investigating GABA Receptor Modulators
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Picrotoxin (50 μM), L-655 708 (11,12,13,13a-Tetrahydro-7-methoxy-9-oxo-9H-imidazo[1,5-a]pyrrolo[2,1 c][1,4]benzodiazepine-1-carboxylic acid, ethyl ester, 10 μM), SCS (Salicylidene salicylhydrazide, 1 μM), TPMPA ((1,2,5,6-Tetrahydropyridin-4-yl) methylphosphinic acid, 50 μM), etomidate (10 μM), allopregnanolone (100 nM), muscimol (5 μM), bumetanide (10 μM), nifedipine (10 μM), benidipine (10 μM), bay K 8644 (10 μM), bicuculline (50 μM), ATP (150 μM; all from Tocris Bioscience, Bristol, UK), SNAP ((S)-Nitroso-N-acetylpenicillamine, 50 μM), SC (semicarbazide 50 μM), GABA (5 μM), geneticin (10 μM; all from Sigma-Aldrich) and CPCPT (1-(3-Chlorophenethyl)−3-cyclopentylpyrimidine-2,4,6-(1H,3H,5H)-trione, 1 μM, Merck Millipore, Darmstadt, Germany) were used at the indicated concentrations, if not differently stated. All pharmacological treatments and live cell stainings were performed in CM at 37°C and 5% CO2.
10
Preparing Chemical Solutions for Research
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All chemicals were purchased from Sigma (Oakville, ON, Canada) except for bumetanide and BAPTA-AM (Tocris, Bioscience, Bristol, UK). Drugs were dissolved in control Ringer’s solution (Table 1 ) or DMSO (max DMSO concentration <0.1%).
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