Hydroxychloroquine

Manufactured by Merck Group

Sourced in United States, Germany

Hydroxychloroquine is a medication primarily used as an antimalarial drug. It is a synthetic derivative of chloroquine, a compound extracted from the bark of the cinchona tree. Hydroxychloroquine is commonly used in the treatment of certain autoimmune disorders, such as rheumatoid arthritis and lupus.

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Lab products found in correlation

Chloroquine, by Merck Group (13 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (11 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (7 mentions) Bafilomycin a1, by Merck Group (6 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (6 mentions) Dimethyl sulfoxide (dmso), by Merck Group (5 mentions) Rapamycin, by Merck Group (4 mentions) Methyl thiazolyl tetrazolium (mtt), by Merck Group (3 mentions) Lipopolysaccharides (lps), by Merck Group (3 mentions) Mg132, by Merck Group (3 mentions) Azithromycin, by Merck Group (3 mentions) Dexamethasone (dex), by Merck Group (3 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (3 mentions) Propidium iodide, by Thermo Fisher Scientific (3 mentions) Antimycin a, by Merck Group (2 mentions) Monensin, by Merck Group (2 mentions) Anti lc3a b, by Cell Signaling Technology (2 mentions) Rifampin, by Merck Group (2 mentions) Ciprofloxacin, by Merck Group (2 mentions) Pam3csk4, by InvivoGen (2 mentions)

62 protocols using hydroxychloroquine

Mitochondrial Depletion and Autophagy Modulation

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All cell lines were cultured in DMEM (Gibco) containing 10% FBS, 4.5 g l⁻¹ glucose, 1 mM l-glutamine, 0.11 g l⁻¹ pyruvate and maintained at 37 °C in 5% CO₂ atmosphere. For mitochondria depletion, cells were treated with 1 μM antimycin A (Sigma A8674) and 1 μM oligomycin (Sigma O4876) every 12 h for the indicated time periods as previously described [18] (link). We recommend that a titration of these drugs is undertaken in different cell systems in order to determine the most effective concentration to cause mitochondrial damage and depletion in any given system.
In order to activate autophagy, cells were treated with 100 nM rapamycin (LCL laboratories R-5000) or 150 μM of deferoxamine mesylate (DFO) for 2 h prior treatment with oligomycin and antimycin A. For the inhibition of the autophagic flux cells were treated with 10 μM hydroxychloroquine (Sigma) or 100 nM bafilomycin A1 (Sigma) together with antimycin A and oligomycin.

Baudot A.D., Haller M., Mrschtik M., Tait S.W, & Ryan K.M. (2015). Using enhanced-mitophagy to measure autophagic flux. Methods (San Diego, Calif.), 75, 105-111.

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Autophagy Protein Detection and Inhibition

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The following primary antibodies were used at the indicated dilutions: anti-LC3A/B (1:1000 for western blotting and 1:100 for immunofluorescence; Cell Signaling Technology, 4108), anti-LC3A (1:100 for immunohistofluorescence; Abgent, 1805a), anti-ATG12 (1:100; Cell Signaling Technology, 2010), anti-ATG13 (1:1000; Cell Signaling Technology, 6940), anti-ATG16L1 (1:1000; Cell Signaling Technology, 8089), anti-human LAMP1 and anti-mouse LAMP1 (1:100; Becton Dickinson, 555798 and 553792), anti-WIPI2 (1:100; Bio-Rad, MCA5780GA), anti-CDSN/corneodesmosin (R&D Systems, AF5725), anti-GAPDH (1:2000; Santa Cruz Biotechnology, sc-25778), and anti-ATP6V0D1 (1:50; ABCAM, ab56441). The following inhibitors and reagents were used: Amiodarone hydrochloride (Sigma, A8423), bafilomycin A₁ (Tocris Biosciences, 1334), betahistine dihydrochloride (Sigma, B4638), chloroquine (Sigma, C6628), CCCP (Sigma, C2759), HBSS (Gibco, 14025–092), hydroxychloroquine (Sigma, H0915), lidocaine (Sigma, L7757), lidocaine hydrochloride monohydrate (Sigma, L5647), monensin (Sigma, M5273), NH₄Cl (Sigma, 213330), nigericin (Sigma, N7143), PP242 (Tocris, 4257), procainamide hydrochloride (Sigma, P9391). Mito-TMRE (abcam, 113852), PIK3C3/VPS34 inhibitor (IN-1) was kindly provided by Dr I. Ganley, University of Dundee.

Jacquin E., Leclerc-Mercier S., Judon C., Blanchard E., Fraitag S, & Florey O. (2017). Pharmacological modulators of autophagy activate a parallel noncanonical pathway driving unconventional LC3 lipidation. Autophagy, 13(5), 854-867.

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Coxiella burnetii Growth and Antibiotic Preparation

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The Coxiella burnetii strain used in this study was Nine Mile Phase 2, clone 4 (NMP2), a low virulence strain that has a large genomic deletion that removes genes responsible for the complexity of LPS side chains^{29 (link)}. Stocks of NMP2 were prepared by growth in ACCM-2 media at pH 4.75, and freezing in sodium phosphate glutamate (SPG) buffer. Antibiotics were purchased as follows: doxycycline hyclate, ciprofloxacin, rifampin, hydroxychloroquine, erythromycin, and levofloxacin (Sigma, St. Louis, MO), azithromycin dihydrate, and moxifloxacin hydrochloride (United States Pharmocopeia, Rockville, MD). doxycycline hyclate, moxifloxacin hydrochloride, ciprofloxacin, and hydroxychloroquine stocks were prepared by dissolving in deionized water at a concentration of 1280 μg/ml. levofloxacin was dissolved in an aqueous solution of 0.05 M NaOH at 1280 μg/ml. erythromycin and azithromycin were dissolved in 95% ethanol at 1280 μg/ml.

Smith C.B., Evavold C, & Kersh G.J. (2019). The Effect of pH on Antibiotic Efficacy against Coxiella burnetii in Axenic Media. Scientific Reports, 9, 18132.

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Quantification of Antimalarial Drugs

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Hydroxy chloroquine and chloroquine as the internal standard were obtained from Sigma Aldrich (United States of America). Whole blood was obtained from Indonesian Red Cross (Jakarta, Indonesia), and volumetric absorptive microsampling was obtained from Neoteryx® (Torrance, CA, United States of America). Methanol and acetonitrile are HPLC grade and were obtained from Merck Co. Ltd. (Darmstadt, German) along with diethylamine, triethylamine, ammonia, n-hexane, and ethyl acetate. Aquabidest used as a solvent was obtained from Ikapharmindo (Jakarta, Indonesia).

Harahap Y., Rohadatul ‘Aisy S.A, & Maggadani B.P. (2021). Development and Validation of the Quantification Method for Hydroxychloroquine in Volumetric Absorptive Microsampling (VAMS) Using High-Performance Liquid Chromatography-Photodiode Array. Advances in Pharmacological and Pharmaceutical Sciences, 2021, 3500279.

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Evaluation of Pharmacological Inhibitors

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T-3764518 (SCD1 inhibitor) [24 (link)] and compound 7a [acetyl-CoA carboxylase (ACC) 1/2 dual inhibitor] [25 (link)] were synthesized by Takeda Pharmaceutical Company. AZD8055 [mammalian target of rapamycin (mTORC) inhibitor] and STA5326 (PIKfyve inhibitor) were purchased from Selleck (Houston, TX, USA). Vacuolin-1 (PIKfyve inhibitor) [26 (link)] was purchased from Merck Millipore (Darmstadt, Germany). GSK2194069 [fatty acid synthase (FASN) inhibitor] was purchased from Chemexpress Co., Ltd. (Shanghai, China). The Bax channel blocker (#2160) was purchased from Tocris (Bristol, UK), and hydroxychloroquine was purchased from Sigma-Aldrich (St. Louis, MO, USA). E64d and pepstatin A were purchased from Peptide Institute Inc. (Osaka, Japan).

Ono A., Sano O., Kazetani K.I., Muraki T., Imamura K., Sumi H., Matsui J, & Iwata H. (2017). Feedback activation of AMPK-mediated autophagy acceleration is a key resistance mechanism against SCD1 inhibitor-induced cell growth inhibition. PLoS ONE, 12(7), e0181243.

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Autophagy Regulation by mTOR-AMPK Pathway

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t-BHP, acridine orange (AO), monodansylcadaverine (MDC), MTT, rapamycin and hydroxy-chloroquine were obtained from Sigma-Aldrich, Merck KGaA. Hoechst 33342 was obtained from Molecular Probes, Thermo Fisher Scientific, Inc. The primary antibody against microtubule-associated protein 1A/1B-light chain 3 (LC3; cat. no. L8918) was purchased from Sigma-Aldrich, Merck KGaA. Primary antibodies against β-actin (cat. no. 4967), p62 (cat. no. 8025), mTOR (cat. no. 2983), 5'-AMP-activated protein kinase (AMPK; cat. no. 5831), unc-51 like autophagy activating kinase 1 (Ulk1; cat. no. 8054), phosphorylated (p)-mTOR (cat. no. 5536), p-AMPK (cat. no. 2535) and p-Ulk1 (cat. no. 14202) were obtained from Cell Signaling Technology, Inc. All primary antibodies were rabbit anti-rat antibodies (dilution, 1:1,000). Anti-rabbit IgG, horseradish peroxidase-conjugated antibody (cat. no. 7074) was purchased from Cell Signaling Technology, Inc. Monomeric red fluorescent protein (mRFP)-green fluorescent protein (GFP)-LC3 adenovirus (cat. no. HB-AP21000001; https://www.hanbio.net/cn/services/item/7) was purchased from Hanbio Biotechnology Co., Ltd. The lactate dehydrogenase (LDH) kit (cat. no. C0016) was obtained from Beyotime Institute of Biotechnology. DMEM and FBS were obtained from Thermo Fisher Scientific, Inc. The purity of DT-010 was >95% (22 (link)).

Xie C., Luo J., Hu H., Wang L., Yu P., Xu L., Sun Y., Wang Y, & Shan L. (2020). A novel danshensu/tetramethypyrazine derivative attenuates oxidative stress-induced autophagy injury via the AMPK-mTOR-Ulk1 signaling pathway in cardiomyocytes. Experimental and Therapeutic Medicine, 21(2), 118.

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Antiviral Drug Treatment Evaluation in Cardiac Myocytes

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Cultures were treated with or without 5 μM chloroquine (Sigma; #C6628) (21 (link)), 5 μM hydroxychloroquine (Sigma; # H0915), 5 μM remdesivir (Biosynth Carbosynth; AG170167), 25 μM lopinavir/ritonavir (Sigma; #SML1222-10MG, #SML0491-10MG), and lopinavir/ritonavir/8 U interferon-β (Sigma; #IF014) for 24 h. After treatment, cells were analyzed as described below. Drug concentrations chosen are based on literature data and are below the 50% cytotoxic concentrations (22 (link), 23 (link)). Incubation of cardiac myocytes with drugs for 24 h under normal culture conditions did not result in cytotoxicity.

Jakobi T., Groß J., Cyganek L, & Doroudgar S. (2022). Transcriptional Effects of Candidate COVID-19 Treatments on Cardiac Myocytes. Frontiers in Cardiovascular Medicine, 9, 844441.

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Modulation of Endo-Lysosomal Trafficking

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If not otherwise indicated, small molecule inhibitors were added to the cells 1 hr before stimulation using the following concentrations: 40 µM MCC950 (HEK293 NLRP3 ASC cells), 5 µM MCC950 (all other cells), 12.5 µM Dynasore, 50nM Bafilomycin A1, 150 nM Thapsigargin, 50 µM Chloroquine, 50 µM Hydroxy-Chloroquine (all Sigma-Aldrich), 20 µM Z-VAD-FMK (Peptide Institute). Of note, the inhibition of endo-lysosmal flux is not typically associated with Thapsigargin, commonly referred to as SERCA inhibitor, or Dynasore, commonly referred to as an inhibitor of dynamin-dependent endocytosis. However, it was recently noted, that Thapsigargin efficiently blocks RAB7-dependent trafficking to the lysosome independently of SERCA inhibition (Ganley et al., 2011 (link)) and that Dynasore also blocks endo-lysosomal flux (Mesaki et al., 2011 (link)). For blocking the efflux of intracellular cells were incubated in medium that was diluted with 135mM KCl (Roth) in sterile water containing 10% FCS to contain indicated concentrations of K⁺.

Gaidt M.M., Ebert T.S., Chauhan D., Ramshorn K., Pinci F., Zuber S., O’Duill F., Schmid-Burgk J.L., Hoss F., Buhmann R., Wittmann G., Latz E., Subklewe M, & Hornung V. (2017). The DNA Inflammasome in Human Myeloid Cells Is Initiated by a STING-Cell Death Program Upstream of NLRP3. Cell, 171(5), 1110-1124.e18.

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Modulation of PBMC Responses by Chloroquine, Hydroxychloroquine, and Rapamycin

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Human PBMCs were trained as described before. In short, 500.000 PBMCs were added into 96-well flat bottom plates. Cells were allowed to adhere for 1h at 37°C. Cells were washed three times with PBS prior to stimulations. After washing cells were incubated with culture medium only as negative control, or treated with 100 μM chloroquine (Sigma Aldrich), 100 μM hydroxychloroquine (Sigma Aldrich) or 0.01 μM rapamycin (Selckchem) for 1 hour at 37°C. The chloroquine and hydroxychloroquine dose were based on a study by French et al.⁴⁹ (link) who showed that in vitro 100 uM is necessary to generate intracellular levels similar to those in patients receiving therapy with hydroxychloroquine 400 mg daily.⁴⁹ (link) Subsequently cells were incubated with 10⁵ cells/ml HKCA (Invivogen) for 24 hours together with the respective treatment for 24 hours at 37°C. Subsequently, cells were washed and cells were rested for five days in RPMI culture medium containing 10% FBS. After the resting period cells were stimulated with either RPMI as negative control, 10ng/ml LPS (Sigma Aldrich) 1ug/ml Pam3CSK4 (Invivogen), 10ug/ml polyI:C (Invivogen), 10ng/ml IFNα (Invivogen), 10ng/ml IFNβ (R&D systems) or 100ng/ml IFNγ (Invivogen). Where indicated hydroxychloroquine and chloroquine were added 1 hour prior to restimulation and 24 hours during restimulation.

Rother N., Yanginlar C., Lindeboom R.G., Bekkering S., van Leent M.M., Buijsers B., Jonkman I., de Graaf M., Baltissen M., Lamers L.A., Riksen N.P., Fayad Z.A., Mulder W.J., Hilbrands L.B., Joosten L.A., Netea M.G., Vermeulen M., van der Vlag J, & Duivenvoorden R. (2020). Hydroxychloroquine Inhibits the Trained Innate Immune Response to Interferons. Cell Reports Medicine, 1(9), 100146.

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Cell Signaling Protein Analysis Protocol

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Transfection reagents Lipofectamine 2000 and Lipofectamine 3000 were purchased from Invitrogen, RNA synthesis inhibitor actinomycin D, protease inhibitor MG132, lysosomal inhibitors bafilomycin and hydroxychloroquine, CDK inhibitor RO-3306, and thymidine were purchased from Sigma-Aldrich. λPPase was purchased from New England BioLabs. Anti-CELF6 (ab173282) and anti-GFP (ab1218) were obtained from Abcam, anti-p21^Waf1/Cip1 (#2947), anti-p27^Kip1(#3686), anti-Gadd45α (#4632), anti-Cyclin B1(#12231), anti-β-TrCP (#4394), anti-Wee1 (#13084), anti-cleaved PARP (#5625), anti-cleaved Casp3 (#9661), anti-cyclin E1 (#20808), and anti-Ubiquitin (#3936) antibodies were purchased from Cell Signaling Technology, anti-P53 (10442-1-AP), anti-GFP (66002-1-1 g), anti-His (66005-1-1 g), anti-β-tubulin (60008-1-1 g), and anti-GAPDH (10494-1-AP) were obtained from Proteintech, anti-LC3B (L7543) was obtained from Sigma-Aldrich.

Liu G., Zhang Q., Xia L., Shi M., Cai J., Zhang H., Li J., Lin G., Xie W., Zhang Y, & Xu N. (2019). RNA-binding protein CELF6 is cell cycle regulated and controls cancer cell proliferation by stabilizing p21. Cell Death & Disease, 10(10), 688.

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