GBAKO THP-1 cells were treated with 1.6 µM recombinant human glucocerebrosidase (rhGCase) imiglucerase as enzyme replacement therapy (Cerezyme; Genzyme, Cambridge, MA, USA). For substrate reduction therapy, cells were treated with 20 µM of D,L-threo-PDMP (Eliglustat—Matreya LLC, State College, PA, USA), a glucosylceramide synthase inhibitor. Ambroxol hydrochloride (A9797, Sigma-Aldrich) and the anti-inflammatory molecule pentosan polysulfate (PPS—Bene pharmaChem, Geretsried, Germany) were used at 100 μM and 5 μg/mL, respectively [45 (link)]. In all cases, cells were treated for 48 and 96 h, as previously described [45 (link),48 (link),50 (link)].
Ambroxol hydrochloride
Manufactured by Merck Group
Sourced in Germany
Ambroxol hydrochloride is a pharmaceutical compound used as an active ingredient in various medications. It is a mucolytic agent that helps thin and loosen mucus, facilitating its clearance from the respiratory tract. The core function of ambroxol hydrochloride is to aid in the management of respiratory conditions, such as bronchitis and chronic obstructive pulmonary disease (COPD), by improving mucociliary clearance.
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Lab products found in correlation
L glutamine, by Merck Group (1 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Sodium phosphate dibasic, by Merck Group (1 mentions)
Formic acid, by Merck Group (1 mentions)
High fat diet (hfd), by Bio-Serv (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Santa Cruz Biotechnology (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Sodium taurocholate hydrate, by Merck Group (1 mentions)
4 methylumbelliferyl β d glucopyranoside, by Merck Group (1 mentions)
Polyclonal rabbit antigoat immunoglobulins hrp, by Agilent Technologies (1 mentions)
Polyclonal goat anti mouse immunoglobulins hrp, by Agilent Technologies (1 mentions)
4 methylumbelliferyl n acetyl β d glucosaminide, by Merck Group (1 mentions)
Diphenhydramine hydrochloride, by Merck Group (1 mentions)
11 protocols using ambroxol hydrochloride
1
Treating Gaucher's Disease with Enzyme Therapy
2
Fibroblast Culture and Ambroxol Treatment
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Collection and use of human tissue were done in agreement with the principles of the Declaration of Helsinki. Fibroblasts were obtained from skin biopsies that were performed at the IRCCS Mondino Foundation under a research protocol previously approved by the institutional Ethic Committee. Informed consent was obtained from all subjects who underwent the procedure. Fibroblast cultures were grown in RPMI-1640 Medium (Sigma Aldrich) with 10% serum (Sigma Aldrich), 2 mM L- glutamine (Sigma Aldrich), 100 μg/ml streptomycin and 100 units/ml penicillin. Analyses were carried out at low culture passages, and disease and control cultures were matched for passage number. In some experiments, fibroblast cultures were treated with ambroxol. Ambroxol hydrochloride (Sigma Aldrich) was dissolved in dimethylsulphoxide (DMSO; 20 mM stock concentration) and diluted in cell culture medium. Cells were treated with 60 μM (final concentration) ambroxol or DMSO for 15 days and, during this treatment time, culture medium was changed every third day. Fresh medium also contained ambroxol or DMSO.
3
Preparation and Characterization of Sodium Hypochlorite
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Commercially available preparations of ambroxol hydrochloride (≥98.0%) and bromhexine hydrochloride (≥98.0%) were purchased from Sigma-Aldrich (Darmstadt, Germany). Formic acid (ACS reagent, ≥96%), sodium chloride (ACS reagent, ≥99.0%) and sodium phosphate dibasic (ACS reagent, ≥99.0%) obtained from Sigma-Aldrich (Darmstadt, Germany), ortho-phosphoric acid (≥87%, Komponent-Reaktiv, Moscow, Russia), methanol (for gradient HPLC, Khimmed, Moscow, Russia) and Type I deionized water with a resistivity of 18.2 MΩ cm−1 were used to prepare the reaction mixtures, mobile phase for HPLC and stock solutions of mucolytic drugs.
Potassium permanganate (chem. pure), sodium hydroxide (chem. pure), hydrochloric acid (35–38%), sodium thiosulfate pentahydrate (for analysis), potassium iodide (chem. pure) and sulfuric acid (93.5–95.6%) purchased from Komponent-Reaktiv (Moscow, Russia) were used to obtain sodium hypochlorite from gaseous chlorine according to the known procedure [41 ], determine the active chlorine content [42 ] and terminate the reaction by quenching the active chlorine with thiosulfate-anions. According to the results of iodometric titration, the content of active chlorine in the prepared hypochlorite solution was 80 g L−1.
Potassium permanganate (chem. pure), sodium hydroxide (chem. pure), hydrochloric acid (35–38%), sodium thiosulfate pentahydrate (for analysis), potassium iodide (chem. pure) and sulfuric acid (93.5–95.6%) purchased from Komponent-Reaktiv (Moscow, Russia) were used to obtain sodium hypochlorite from gaseous chlorine according to the known procedure [41 ], determine the active chlorine content [42 ] and terminate the reaction by quenching the active chlorine with thiosulfate-anions. According to the results of iodometric titration, the content of active chlorine in the prepared hypochlorite solution was 80 g L−1.
4
Ambroxol Hydrochloride Treatment for ICH
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Ambroxol hydrochloride was purchased from Sigma-Aldrich (St. Louis, MO) and dissolved in saline. For treatment groups, mice intraperitoneally (IP) received 35 mg/kg or 70 mg/kg Ambroxol hydrochloride immediately and 24 h after ICH, followed by once a day for 2 days. For the ICH group, mice received an equal volume of saline at the same time point as treatment groups. In the sham group, mouse received saline only.
5
Modulating Lysosomal Enzyme NEU1 Regulates Metabolic Health
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Approval for the animal care and the use in the experiments was granted by the Animal Care and Use Committee of CHU Ste-Justine. C57BL/6J WT mice and previously described CathAS190A−Neo mice with ∼90% reduction of NEU1 activity [31] (link) were maintained on a 12 h dark/light cycle. Four-month-old (∼25 g BW) male mice received ad libitum water and either normal diet (5% fat, 57% carbohydrate) or HFD (35% fat, 36% carbohydrate, Bio-Serv). Body weight and total food consumption were measured. In the first study, after 7 weeks on HFD, NEU1 activity was induced pharmacologically in mice by intraperitoneal injection of Ambroxol hydrochloride (Sigma) at the dose of 60 mg/kg body weight/mouse for 5 consecutive days. In the second study, 4-month-old mice were fed with HFD and injected one day later through the tail vein with adenovirus expressing CathA-IRES-NEU1-GFP or control NEU1-GFP at a dose of 109 TU/mouse in 0.2 ml of saline. These mice were fed with a HFD continuously before sacrifice 20 weeks later. Intraperitoneal Glucose Tolerance Test (IGTT) and Insulin Tolerance Test (ITT) were conducted as described [16] (link).
6
Ambroxol Hydrochloride Tablet Formulation and Characterization
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Flavamed® packaging of tablets was provided by the international research-based pharmaceutical company Berlin-Chemie AG, Berlin, Germany (Menarini Group), Glienicker Weg 125, 12489 Berlin, Germany. Each tablet contains 30 mg of Ambroxol hydrochloride (AMB). The external appearance of the tablet: white, round tablets with flat surfaces and faceted edges, with an embedded dividing line on one side. In addition to the active (drug) ingredient, the other excipients are as follows: (a) lactose monohydrate (filler in matrix tablet), (b) corn starch (disintegrant and binder in matrix tablet), (c) powdered cellulose (disintegrant in matrix tablet), (d) croscarmellose sodium (CCS) (FDA (U.S.—Food and Drug Administration) approved superdisintegrant), (e) povidone (polyvinylpyrrolidone, PVP) K30 (binder in matrix tablet) and (f) magnesium stearate (lubricant in matrix tablet). Ambroxol hydrochloride, a reference pharmaceutical standard, was purchased from Sigma-Aldrich, St. Louis, MO, USA.
7
Culturing Fibroblasts for Gaucher's Disease
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Fibroblasts from two patients affected by type-3 GD were cultured and maintained in Dulbecco's modified Eagle's medium High Glucose (EuroClone) containing 10% fetal bovine serum (Gibco), 1% glutamine (Gibco), and 1% penicillin/streptomycin (Gibco), in a humidified atmosphere containing 5% CO2 at 37 °C.
Fibroblasts, grown on 100 mm plates, were treated with Ambroxol hydrochloride (A9797, Sigma-Aldrich) 100 μM dissolved in Dimethyl Sulfoxide 0,1% (DMSO – Santa Cruz Biotechnology) or with an equal volume of vehicle (DMSO), as control [6 (link)]. Twenty-two hours later, cells were lysed in water and sonicated. Total amount of protein in cell lysates was determined by Bradford assay, using the Biorad-Protein Assay (BioRad), following manufacturer's instructions.
Fibroblasts, grown on 100 mm plates, were treated with Ambroxol hydrochloride (A9797, Sigma-Aldrich) 100 μM dissolved in Dimethyl Sulfoxide 0,1% (DMSO – Santa Cruz Biotechnology) or with an equal volume of vehicle (DMSO), as control [6 (link)]. Twenty-two hours later, cells were lysed in water and sonicated. Total amount of protein in cell lysates was determined by Bradford assay, using the Biorad-Protein Assay (BioRad), following manufacturer's instructions.
8
Biochemical Analysis of α-Synuclein Pathways
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4‐Methylumbelliferyl β‐D‐glucopyranoside, 4‐methylumbelliferyl N‐acetyl‐β‐D‐glucosaminide, sodium taurocholate hydrate, and ambroxol hydrochloride were purchased from Sigma‐Aldrich (St Louis, MO). Pierce BCA Protein Assay, Halt Protease Inhibitor Cocktail, Halt Phosphatase Inhibitor, Pierce ECL Western Blotting Substrate, and Power SYBR Green PCR Master Mix were purchased from Thermo Scientific (Waltham, MA). Luminata Forte Western HPR Substrate was purchased from Millipore (Billerica, MA). RNeasy Mini Kit was purchased from Qiagen (Hilden, Germany). Precision nanoScript 2 Reverse Transcription kit (RT‐nano‐Script2) was purchased from Primerdesign (Chandler's Ford, UK). Anti–α‐synuclein antibody (4D6; ab1903), anti–α‐synuclein (phospho S129) antibody (EP1536Y; ab51253), anti–mitochondrial transcription factor A (TFAM) antibody (ab131607), and anti–transcription factor EB (TFEB) antibody ‐ ChIP grade (ab2636) were purchased from Abcam Biochemicals (Cambridge, UK). Polyclonal swine antirabbit immunoglobulins/horseradish peroxidase (HRP), polyclonal goat antimouse immunoglobulins/HRP, and polyclonal rabbit antigoat immunoglobulins/HRP were purchased from Dako (Glostrup, Denmark).
9
Ambroxol Titration for Gaucher Disease
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An initial titration experiment was performed to select the dose of Ambroxol hydrochloride to use for rescue experiments. Ambroxol hydrochloride (Sigma) was dissolved in dimethylsulphoxide (DMSO) and diluted in cell culture media to give a final concentration of 10 µM, 30 µM and 60 µM. This dose range was selected based upon dosages used in previous in vitro studies (Maegawa et al., 2009 (link); Luan et al., 2013 (link)). In the initial titration experiment a control line and a Gaucher disease line were treated with 10 µM, 30 µM and 60 µM for 5 days. Media were changed daily for ambroxol treated and control lines. Controls were treated with DMSO only. The greatest increase in glucosylceramidase protein levels were observed with 60 µM (Supplementary Fig. 1 ), and this dose was therefore selected for the rescue experiments.
10
Ambroxol Treatment for Neuronal Cultures
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Ambroxol hydrochloride (Sigma) was prepared in DMSO at a concentration of 10 mM, in order to be administered to neurons in culture at a concentration of 10, 30 and 60 µM. At day 4 or 5 in culture neuronal media was changed to fresh neuronal media with the different doses of ambroxol. 3 days after the first treatment with ambroxol, the neurons were treated again for an extra 2 days. After 5 days of ambroxol treatment neurons were harvested and assays performed.
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