6–12 week old female C57Bl/c mice were obtained from Jackson Laboratories. Mice received intraperitoneal injections 3 times every 12 h with clioquinol, amantadine (both at 30 mg/kg per dose, Sigma Aldrich) or appropriate controls. Dosing level and frequency were chosen based on previous experiments using clioquinol in mice47 (link),68 and the drug half-life (11–14 h). clioquinol was dissolved in 8% PEG400/PBS heated to 37 °C; amantadine was dissolved in PBS. Before injection, the solutions were shaken several times. Mice were sacrificed for tissue collection between 2.5 and 3 h after the last treatment. Blood collection was obtained from the tail. For guanfacine treatment, mice received initial injection of 5 mg/kg of drug or vehicle control (PBS) on day 1, followed by two (experiment 1) or six (experiments 2–5) intraperitoneal injections at 2 mg/kg every 12 h starting on day 2. Mice were sacrificed for tissue collection 12 h after the last treatment. All animal procedures were done according to protocols approved by the Mount Sinai School of Medicine Institutional Animal Care and Use Committee.
Clioquinol
Manufactured by Merck Group
Sourced in United States
Clioquinol is a pharmaceutical compound used as a laboratory reagent. It is a quinoline-based compound with antimicrobial properties. Clioquinol is commonly utilized in various in vitro and in vivo research applications, but its specific core function and intended uses should not be extrapolated upon in this response.
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Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Amantadine, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Formic acid, by Merck Group (2 mentions)
Clbq14, by Tokyo Chemical Industry (2 mentions)
Disulfiram, by Merck Group (2 mentions)
Nitroxoline, by Merck Group (2 mentions)
Fitc dextran, by Merck Group (1 mentions)
Lysotracker red probe, by Thermo Fisher Scientific (1 mentions)
Human serum albumin (hsa), by Merck Group (1 mentions)
Donepezil, by Merck Group (1 mentions)
1 3 dimethylaminopropyl 3 ethylcarbodiimide hydrochloride, by Merck Group (1 mentions)
Aβ1 42, by GL Biochem (1 mentions)
Tat peptide, by GL Biochem (1 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Butylated hydroxyanisole, by Merck Group (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Alpha mem, by Thermo Fisher Scientific (1 mentions)
Anti mouse igg hrp, by Merck Group (1 mentions)
12 protocols using clioquinol
1
Intraperitoneal Injection of Clioquinol, Amantadine, and Guanfacine in Mice
2
Protein Conjugation and Cellular Uptake Assay
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Donepezil, clioquinol, HSA, ThT, 1‐(3‐dimethylaminopropyl)‐3‐ethylcarbodiimide hydrochloride (EDC), N‐hydroxysuccinimide (1‐hydroxypyrrolidine‐2,5‐dione, NHS), and FITC‐dextran were purchased from Sigma–Aldrich; GM1 was purchased from J&K(Beijing); TAT peptide and Aβ1‐42 were purchased from GL Biochem (Shanghai), and the purity of the peptides was greater than 98%; glutaric dialdehyde was purchased from Aladdin (Shanghai); hexafluoroisopropanol (HFIP) and 4‐(N‐maleimidomethyl) cyclohexane‐1‐carboxylic acid 3‐sulfo‐N‐hydroxysuccinimide ester sodium salt (Sulfo‐SMCC) were purchased from Yuanye (Shanghai). FITC‐transferrin, FITC‐cholera toxin subunit B, and LysoTracker Red probe were purchased from Invitrogen (USA). Milli‐Q water (18 MΩ cm) was used in the experiments. Nicotinic acetylcholine receptors α7(α7 nAChR), SYAP1 and GAP43 antibody were purchased from Bioss (Beijing). Rat cysteine protease‐3 ELISA kit was purchased from Huyu Biotechnology (Shanghai).
3
Stem Cell Differentiation Assay Protocol
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Fetal bovine serum (FBS), DMEM, alpha MEM, gentamycin, phosphate-buffered saline (PBS), and 0.25% Trypsin EDTA were purchased from Invitrogen (CA, USA). 2-Mercaptoethanol (BME), zinc chloride (ZnCl2), clioquinol (CQ), calcium disodium ethylene diamine tetraacetate (CaEDTA), Triton X-100, dimethylsulfoxide (DMSO), butylated hydroxyanisole (BHA), anti-mouse IgG-HRP, and anti-rabbit IgG-HRP were purchased from Sigma (MO, USA). Anti-TUJ-1 and Alexa 488 were purchased from Abcam (MA, USA). Anti-phospho-ERK1/2, Anti-ERK1/2, and skim milk were purchased from Santa Cruz (CA, USA). Alexa Fluor 647 anti-human CD90 and FITC anti-human CD105 were purchased from BioLegend (CA, USA). PVDF membrane and ECL were purchased from Bio-Rad (CA, USA). PMSF and protease inhibitor were purchased from Roche (Mannheim, Germany). M-PER mammalian protein extraction reagent was purchased from Thermo Fisher Scientific (IL, USA). CCK-8 was purchased from Dojindo Laboratories (Kumamoto, Japan). FGF-basic (bFGF) was purchased from PeproTech (CA, USA). Bovine serum albumin (BSA) was purchased from BioShop (CA, USA).
4
Biopharmaceutical Characterization Protocol
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CLBQ14 (purity ≥ 98%) was purchased from TCI Chemicals (Tokyo, Japan). LC-MS grade water and acetonitrile, clioquinol, formic acid, trifluoroacetic acid (TFA), ethanol, Tween 20, Tween 80, soybean oil, paraffin oil, olive oil, dimethyl sulfoxide (DMSO), N’N dimethyl acetamide (DMA), polyethylene glycol 400 (PEG 400), glycerol, 1-octanol, 0.85% sodium chloride solution, phosphate buffered saline tablets, and CD-1 mouse, Sprague Dawley (SD) rat, cynomolgus monkey and human microsomes were purchased from Sigma Aldrich (St. Louis, MO). Transcutol High Purity (HP)®, Labrasol® and Capyrol 90® were gifts from Gattefosse (Lyon, France). Recombinant human cytochrome P450 enzymes (rhCYP) were purchased from BD Biosciences (San Jose, CA). Heparin (1000 units/mL) and pharmaceutical grade normal saline were purchased from Hospira (Lake Forest, IL). Human plasma was purchased from Gulf Coast Blood Center (Houston, TX) and fresh rat plasma was collected from male SD rats (Envigo RMS Inc., Indianapolis, IN) and stored at −80°C until use. CD-1 mouse and cynomolgus monkey plasma were purchased from BioIVT (Westbury, NY). All chemicals and reagents were used as received.
5
Copper-Ionophore Treatment of Prostate Cancer
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TRAMP-C1 mouse prostate cancer cells were derived from the TRAMP (transgenic adenocarcinoma of mouse prostate) strain (C57BL/6 background) [38 (link)] and were generously provided by Assoc. Prof. Michael H. Kershaw (Peter MacCallum Cancer Centre, Melbourne, Australia). Mouse (C57BL/6) primary prostate epithelial cells (PrECs) were purchased from Cell Biologics (Chicago, USA; Cat#C57-6038). TRAMP-C1 cells were cultured in DMEM (ThermoFisher, Scoresby, Australia; Cat#11965-092) supplemented with 10% foetal calf serum (Bovogen biologicals, Keilor East, Australia; Cat#SFBS-F), 2 mM L-glutamine, 100 Units/mL penicillin and 100 μg/mL streptomycin. Primary prostate epithelial cells were cultured in Epithelial Cell Medium (Cell Biologics, Chicago, USA; Cat# M6621) as per the manufacturer's instructions. Cells were maintained at 37°C under humidified atmosphere containing 5% CO2.
Copper-ionophores (Disulfiram; Cat#86720 and clioquinol; Cat#24880) were purchased from Sigma-Aldrich (Castle Hill, Australia) or synthesized [CuII(gtsm)] by Assoc. Prof. Paul S. Donnelly (University of Melbourne, Melbourne, Australia) following published procedures [70 ]. Each copper-ionophore was prepared in DMSO at 5 mM immediately before each experiment. All other reagents were supplied by Sigma-Aldrich (Castle Hill, Australia) unless specified otherwise.
Copper-ionophores (Disulfiram; Cat#86720 and clioquinol; Cat#24880) were purchased from Sigma-Aldrich (Castle Hill, Australia) or synthesized [CuII(gtsm)] by Assoc. Prof. Paul S. Donnelly (University of Melbourne, Melbourne, Australia) following published procedures [70 ]. Each copper-ionophore was prepared in DMSO at 5 mM immediately before each experiment. All other reagents were supplied by Sigma-Aldrich (Castle Hill, Australia) unless specified otherwise.
6
Intraperitoneal Injection of Clioquinol, Amantadine, and Guanfacine in Mice
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6–12 week old female C57Bl/c mice were obtained from Jackson Laboratories. Mice received intraperitoneal injections 3 times every 12 h with clioquinol, amantadine (both at 30 mg/kg per dose, Sigma Aldrich) or appropriate controls. Dosing level and frequency were chosen based on previous experiments using clioquinol in mice47 (link),68 and the drug half-life (11–14 h). clioquinol was dissolved in 8% PEG400/PBS heated to 37 °C; amantadine was dissolved in PBS. Before injection, the solutions were shaken several times. Mice were sacrificed for tissue collection between 2.5 and 3 h after the last treatment. Blood collection was obtained from the tail. For guanfacine treatment, mice received initial injection of 5 mg/kg of drug or vehicle control (PBS) on day 1, followed by two (experiment 1) or six (experiments 2–5) intraperitoneal injections at 2 mg/kg every 12 h starting on day 2. Mice were sacrificed for tissue collection 12 h after the last treatment. All animal procedures were done according to protocols approved by the Mount Sinai School of Medicine Institutional Animal Care and Use Committee.
7
Quantitative Analysis of CLBQ14
8
Clioquinol and Zinc Chloride Protocol
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Analytical grade clioquinol and zinc chloride were purchased from Sigma Chemical Co (Sigma Chemical Co, St. Louis, MO). CQ was dissolved in dimethylsulfoxide (DMSO, Solon, OH) to 10 mmol/L as stock solution (− 20 °C storage) and was diluted by MEM to a final concentration. zinc chloride was dissolved in deionized water to 1 mmol/L as a stock solution (− 20 °C storage). The proteasome inhibitor MG132 was obtained from EMD Biosciences Inc. (San Diego, CA, USA). Antibodies against ATM, γ-H2AX, p65 and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The pNF-κB-Luc reporter construct was from BD Biosciences Clontech (Palo Alto, CA, USA), and the luciferase reporter assay kit was purchased from Promega (Madison, WI, USA).
9
Maintenance of Leukemia Cell Lines
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MM (LP1, RPMI-8226 and OPM2) and leukemia (OCI-AML2, K562 and THP-1) cell lines were maintained in Iscove's modified Dulbecco's medium and RPMI-1640 medium (Invitrogen, CA, USA), respectively, as described previously26 (link). Clioquinol, chloroquine, nitroxoline, 3-MA, and BafA1 were obtained from Sigma-Aldrich (St. Louis, MO, USA).
10
Preparation of Quinoline Derivatives
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Clioquinol (1 ), 8-hydroxy-5-quinolinesulfonic acid (2 ), and 8-hydroxy-7-iodo-5-quinolinesulfonic acid (3 ) were purchased from Sigma-Aldrich (St. Louis, MO, USA). These compounds were dissolved in DMSO (Sigma-Aldrich) and diluted in a medium assay to obtain a maximum concentration of 2% DMSO in the experiments.
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