T. b. brucei strain Lister 427 (427-WT) was used as the standard drug-sensitive (wild-type; WT) laboratory strain [34 (link)]. This strain was previously adapted for multi-drug resistance by deletion of the TbAT1/P2 drug transporter [35 (link)], subsequently followed by in vitro exposure to increasing concentrations of pentamidine [28 (link)]. For T. congolense, the culture-adapted Savannah strain IL3000 was used [22 (link)], as well as a clonal line, 6C3, adapted from IL3000 in vitro to diminazene aceturate (Sigma), leading to a ~10-fold resistance. All Trypanosoma strains were cultured and used as bloodstream forms; T. b. brucei in a full HMI-9 medium supplemented with 10% Foetal Bovine Serum (FBS) [27 (link)] and T. congolense in TC-BSF1 medium with 20% goat serum at 34 °C, as described by Coustou et al. [36 (link)]. The promastigotes of L. mexicana strain MNY/BZ/62/M379 were cultured at 25 °C in a minimal essential medium, HO-MEMO-MEME, supplemented with 10% FBS and 1% of a penicillin/streptomycin solution (Gibco), as described previously [37 (link),38 (link)].
Diminazene aceturate
Manufactured by Merck Group
Sourced in United Kingdom, Austria, United States
Diminazene aceturate is a laboratory chemical used as a reference standard. It is a solid crystalline compound with a specific molecular formula and structure. The primary function of diminazene aceturate is to serve as a reference point for analytical and research purposes, enabling accurate identification and quantification of related substances.
Automatically generated - may contain errors
Lab products found in correlation
Prism 5, by GraphPad (3 mentions)
Chloroquine, by Merck Group (2 mentions)
Pentamidine, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulphoxide, by Merck Group (1 mentions)
Sodium phosphate dibasic na2hpo4, by Merck Group (1 mentions)
Penicillin streptomycin l glutamine psg, by Merck Group (1 mentions)
2 4 2 hydroxyethyl piperazin 1 yl ethanesulfonic acid hepes, by Merck Group (1 mentions)
Sybr green 1, by Merck Group (1 mentions)
Sodium bicarbonate, by Merck Group (1 mentions)
Sodium citrate, by Merck Group (1 mentions)
Adenine, by Merck Group (1 mentions)
Phenylarsine oxide, by Merck Group (1 mentions)
Tubercidin, by Merck Group (1 mentions)
Cordycepin, by Merck Group (1 mentions)
Eflornithine, by Sanofi (1 mentions)
Suramin, by Bayer (1 mentions)
Melarsoprol, by Sanofi (1 mentions)
17 protocols using diminazene aceturate
1
Trypanosoma and Leishmania Culture Protocols
2
Cytotoxicity Assay for Antiparasitic Compounds
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HMI9, FBS, and DMEM were purchased from Thermo Fisher Scientific, UK. Other chemicals and reagents such as penicillin-streptomycin-L-glutamine (PSG), 2-[4-(2-hydroxyethyl) piperazin-1-yl] ethanesulfonic acid (HEPES), chloroquine, diminazene aceturate, Alamar dye, SYBR Green I, dimethyl sulphoxide (DMSO), sodium citrate, adenine, sodium bicarbonate (NaHCO3), sodium chloride (NaCl), sodium phosphate dibasic (Na2HPO4), potassium chloride (KCl), sodium phosphate monobasic (KH2PO4), sodium hydroxide (NaOH), and sodium bicarbonate (NaHCO3) were also purchased from Sigma-Aldrich, UK.
3
Histamine Measurement in Murine Plasma
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Citrate plasma containing 10 µM diminazene-aceturate (D7770, Sigma-Aldrich, Austria) was used to measure histamine concentrations using the histamine homogeneous time resolved fluorescence (HTRF) dynamic kit (62HTMDPET, Cisbio, France). The kit was used according to the instructions provided by the manufacturer. A histamine standard curve in pooled plasma of C57Bl/6J mice was used for quantification. All histamine concentrations refer to the histamine base and all measurements were performed in duplicate.
4
Evaluating Antitrypanosomal Compounds
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T. b. brucei were seeded at a density of 3 × 105 cells/mL in 96-well plates in a two-fold dilution of compounds and incubated for 24 h. Normal mouse macrophages (RAW 264.7) were plated at a density of 3 × 105 cells/mL for 48 h to allow for adherence to plates before compounds were added to cells in a two-fold dilution and incubated for another 24 h. The alamarBlue dye (10% v/v) was added to wells and incubated for another 24 h. Experiments were run in triplicates. Spectrophotometric absorbance was recorded at a wavelength of 570 nm. Diminazene aceturate (Sigma-Aldrich, Kent, UK) a known antitrypanosomal drug, was used as a positive control.
5
DIZE Administration in Post-Radiation Injury
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Diminazene aceturate (DIZE) was purchased from Sigma-Aldrich (St. Louis, Missouri, catalog #D7770) and MedChemExpress (catalog #HY-12404, Monmouth Junction, NJ) and reconstituted in sterile water at a stock concentration of 10 mg/ml. Rats were weighed immediately prior to drug administration and administered 15 mg/kg of the 10 mg/ml DIZE solution via subcutaneous injection (Qi et al., 2013 (link)). Vehicle control rats received subcutaneous injections of an equivalent volume of sterile water. Rats were dosed subcutaneously starting at 72 h post-PBI with either 15 mg/kg DIZE or sterile water (3X/week (MWF) for the duration of the study.
6
Measuring Histamine Degradation Kinetics
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Histamine degradation rates were determined by incubating serum or plasma samples with 100 ng/mL (900 nmol/L) histamine base for various durations at 37°C. There is no obvious difference using plasma or serum. The reactions were stopped at different time points by adding a final concentration of 50 μmol/L diminazene aceturate (Sigma, D7770, Vienna, Austria). Diminazene is a potent and selective DAO inhibitor with an inhibition constant Ki of 13 nmol/L against rhDAO.44 The samples were then immediately snap‐frozen in liquid nitrogen and stored at −30°C until measurement of histamine concentration using LC‐MS/MS and ELISA.
7
High-throughput Drug Screening for Trypanosoma
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For all the STIB900 lines drug sensitivities were determined with the Alamar blue assay [27 (link)]. T. b. brucei B48 drug sensitivities were determined as described using a slightly modified protocol [28 (link)]. The SoftMax Pro software and Prism 5 (GraphPad) were used to calculate 50 % inhibitory concentrations (IC50) by non-linear regression fitting to a sigmoidal dose–response curve. All assays were performed at least three times independently, each in duplicate or triplicate. The following compounds were tested, each individually: melarsoprol (Sanofi-Aventis/WHO), eflornithine (Sanofi-Aventis), DB75 (Immtech), fexinidazole (DNDi, Geneva), nifurtimox (WHO, Geneva), suramin (Bayer), pentamidine isethionate, diminazene aceturate, phenylarsine oxide, aminopurinol, cordycepin, adenosine arabinoside, and tubercidin (Sigma). Statistical tests were performed with GraphPad Prism 5.0.
8
Monosodium iodoacetate-induced Osteoarthritis
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Monosodium iodoacetate (Sigma-Aldrich, St. Louis. MO, USA, cat no. I2512), Diminazene aceturate (Sigma-Aldrich, St. Louis, MO, USA) and ketamine/xylazine (Biochemie GmbH, Vienna, Austria) were purchased from commercial suppliers. Losartan was a gift from Amriya for Pharmaceutical industries, Alexandria, Egypt. All drugs were dissolved in saline.
9
Antitrypanosomal Cytotoxicity Screening
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Trypanosome cells were seeded at a density of 1.5 × 105 cells/ml in 96-well plates in a two-fold dilution of fractions and incubated for 24 hours. Normal mouse macrophages (RAW 264.7) were seeded at a density of 1.5 × 105 cells/mL for 48 hours before treatment with fractions in a two-fold dilution and subsequent incubation for another 24 hours. Resazurin (10% V/V) was added to the wells and incubated for another 24 hours. Analysis was conducted in quadruplicates. Spectrophotometric absorbance was measured at a wavelength of 570 nm. Diminazene aceturate (Sigma-Aldrich), a known antitrypanosomal drug, was used as a reference drug.
10
Preparation of HDAC Inhibitor Compounds
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Suberoylanilide hydroxamic acid (SAHA; Sigma Aldrich, USA) and chloroquine (chloroquine diphosphate salt; Sigma Aldrich, USA) were prepared as 10 mM stock solutions in 100% DMSO. Belinostat (PXD101), romidepsin (FK228) and panobinostat (LBH589) were purchased from Selleck Chemicals and prepared as 20 mM stock solutions in 100% DMSO. Pentamidine, diminazene aceturate and puromycin were all purchased from Sigma Aldrich and 0.16, 9.7 and 9.2 mM stock solutions prepared in 100% DMSO.
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