Quiescent parasites were generated using the modified Teuscher, et al. protocol as described by Breglio, et al.22 (link),30 . Briefly, synchronized P. falciparum cultures were treated with 700 nM dihydroartemisinin (Sigma Aldrich, St Louis, MO) in DMSO for 6 h. Cells were washed three times with RPMI to remove drug before being resuspended in media and returned to culture conditions. Cultures were then treated with D-sorbitol every 24 hours for the following 72 hours to remove any actively growing parasites that did not enter quiescence. Immediately following the final sorbitol treatment, flasks were split into two aliquots. One aliquot was left unenriched to serve as the control, while the second aliquot was SLOPE enriched with 55 SLO units as described above. A portion of both untreated and SLOPE samples was then stained with SYBR-Green and MitoProbe DiIC1(5) as described above for flow cytometry analysis. The remainder of each untreated and SLOPE sample was stained for fluorescence microscopy as described below.
Dihydroartemisinin
Manufactured by Merck Group
Sourced in United States
Dihydroartemisinin is a synthetic compound used in laboratory settings. It functions as a precursor for the production of antimalarial drugs.
Automatically generated - may contain errors
Lab products found in correlation
Artemisinin, by Merck Group (5 mentions)
Chloroquine, by Merck Group (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Mefloquine, by Merck Group (2 mentions)
Atovaquone, by Merck Group (2 mentions)
Sodium orthovanadate, by Merck Group (2 mentions)
Amodiaquine dihydrochloride dihydrate, by Merck Group (2 mentions)
Artesunate, by Merck Group (2 mentions)
Proguanil, by Merck Group (1 mentions)
L dihydroorotic acid, by Merck Group (1 mentions)
Antimycin a, by Merck Group (1 mentions)
Amodiaquine, by Merck Group (1 mentions)
Artemether, by Merck Group (1 mentions)
Hypoxanthine, by Merck Group (1 mentions)
Human ab serum, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Methanol, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Saponin, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
13 protocols using dihydroartemisinin
1
Generating Quiescent Malaria Parasites
2
Antimalarial Compound Screening Protocol
3
Anti-malarial Compounds Characterization
4
Western Blot Reagents and Buffers
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4-(2-Hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES) buffer and phosphate buffered saline (PBS), sodium ortho-vanadate, d -glucose, sodium bicarbonate, sodium chloride, sodium fluoride, hypoxanthine, l -glutamine, methanol, DAPI, dihydroartemisinin, and saponin were purchased from Merck-Sigma. RPMI 1640 medium and AB human serum were purchased from Gibco (Carlsbad, CA, USA). RIPA buffer was prepared according to [32 (link)].
5
Investigating JARID2 Regulation in Cells
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Library of Natural Products (Cat No.L1400Selleck Chemicals). Dihydroartemisinin (D7439), docetaxel (01885) from Sigma-Aldrich, MG132 (474791), Calbiochem San Diego, CA, USA) were dissolved in DMSO (Sigma-Aldrich). siRNA-JARID2 (sc-60872, Santa Cruz Biotechnology) was used following the manufacturer’s recommendation.
6
Artemisinin Compound Screening Protocol
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Dihydroartemisinin (DHA), DTT, DMSO, CHX, WR99210 (WR), D-(+)-GlcN were from Sigma-Aldrich; SYTO-61 was from Life Technologies; Protease Inhibitor Cocktail (PIC) was from Roche; epoxomicin was from Sapphire Bioscience or Sigma; b-AP15 was from Calbiochem; RA190 was from Calbiochem; Compound 1 (C1) was a kind gift of Dr Larry Dick, Takeda Pharmaceuticals, and MLN4924 was from MedChemExpress. Shield-1 ligand was kindly provided by Dr Dean Goodman and Prof Geoff McFadden, University of Melbourne, or purchased from Cheminpharma.
7
Antimalarial Drug Evaluation Protocol
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Artemisinin, mefloquine hydrochloride, amodiaquine dihydrochloride dihydrate, artesunate, dihydroArtemisinin, sodium orthovanadate, rhodamine123 (rh123) and rifampicin were purchased from Sigma Aldrich (St Louis, MO, USA). Methylene blue zinc chloride double salt was purchased from Fluka Sigma Aldrich (Steinem, Switzerland). Artemisone was kindly donated by Professor Richard Haynes, The Hong Kong University of Science and Technology, Hong Kong. Vinblastine hydrochloride was purchased from ICN biochemical (Seven hills, NSW, Australia). PSC 833 was donated from Novartis BioPharma (Basel, Switzerland). GF 120918 was donated from Glaxo-SmithKline (Boronia, Vic, Australia) and MK571 was supplied by BIOMOL international (Plymouth meeting, Philadelphia). Mini Protease inhibitor tablets were supplied by Thermo Scientific (Rockford, USA).
8
Synthesis of Malaria Compounds
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MB14 and MB10 were synthesised by R. Kalhor, M. Buskes and B. Abbott as described previously24 (link). The same authors provided RK18 and the method of its synthesis is currently being prepared for publication. Cipargamin was kindly provided by the Medicines for Malaria Venture. Furosemide, glucosamine, artemisinin and dihydroartemisinin were from Sigma.
9
Malaria Parasite Culture and Antimalarial Drug Screening
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Parasites (P. berghei ANKA and P. falciparum 3D7 and Dd2 strains) were obtained from Malaria Research and Reference Reagent Resource Centre (MR4). Cell culture reagents were purchased from Thermo Fisher Scientific and Lonza. YOYO-1 and SYBR green 1 were from Thermo Fisher Scientific. Antimalarials (chloroquine, dihydroartemisinin, and pyrimethamine) and DMSO were from Sigma. Bazedoxifene was from Adooq Bioscience or Cayman Chemical, and tamoxifen and raloxifene were from Adooq Bioscience. All routinely used chemicals and plasticware were purchased from standard companies, such as MP Biomedicals, Sigma, Thermo Fisher Scientific, and Corning. Human blood was collected and processed according to the protocols approved by the Institutional Ethics Committee of Centre for Cellular and Molecular Biology (IEC-38-R2/2015 and IEC-38-R3/2015), and the studies reported herein abided by the declaration of Helsinki principles.
10
In vitro and Animal Studies of Antimalarial Drugs
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In vitro studies. The antimalarial artesunate-amodiaquine (ASAQ Winthrop) (Sanofi-Aventis, Gentilly Cedex, France) was solubilized by crushing the tablets and resuspending in dimethyl sulfoxide to prepare a solution with the AQ component at a concentration of 100 mM. The compounds, amodiaquine dihydrochloride dihydrate (#A2799), artesunate (#A3731), and dihydroartemisinin (#D7439) were purchased from Sigma-Aldrich (Saint Louis, MO). N-desethylamodiaquine hydrochloride (#Sc-212178) was obtained from Santa Cruz Biotechnology (Dallas, TX). Drugs were prepared as 100 mM stocks in dimethyl sulfoxide. Animal studies. Source, formulation, and preparation of amodiaquine hydrochloride was the same for the PK and the efficacy studies. Amodiaquine hydrochloride was obtained from US Pharmacopeia (Rockville, MD; Cat. 1031004; Lot J01144) and a 4 mg/mL AQ (free base) solution was prepared in Sterile Water for Injection (USP).
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