Alpha-Minimal Essential Medium (α-MEM), Dulbecco's modified Eagle's medium F12, and Earle's Balanced Salt Solution cell culture media were obtained from Invitrogen. Fetal calf serum was purchased from Internegocios S.A. Kanamycin (50 μg/ml), chloramphenicol (20 μg/ml), and ampicillin (100 μg/ml); MTT, firefly luciferase, suramin, PPADS, 8-phenyl theophylline, NF110, HK, apyrase, ATP, ADP, AMP, UTP, and suramin were purchased from Sigma-Aldrich. LIVE/DEAD BacLight Bacterial Viability kit and 4',6-diamidino-2-phenylindole were purchased from Molecular Probes.
Suramin
Manufactured by Merck Group
Sourced in United States, Germany, Australia, Japan
Suramin is a laboratory chemical compound that functions as an inhibitor of various enzymes and biological processes. It is commonly used in research applications to study the mechanisms and effects of enzyme inhibition. Suramin exhibits a broad range of biological activities, making it a versatile tool for scientific investigations.
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Lab products found in correlation
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Glutamax, by Thermo Fisher Scientific (5 mentions)
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Carbenoxolone, by Merck Group (4 mentions)
Phosphate buffered saline (pbs), by Merck Group (4 mentions)
Fluo 4 am, by Thermo Fisher Scientific (4 mentions)
Arl67156, by Merck Group (3 mentions)
Ppads, by Bio-Techne (3 mentions)
Thrombin, by Merck Group (3 mentions)
L glutamine, by Merck Group (3 mentions)
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Nifedipine, by Merck Group (3 mentions)
116 protocols using suramin
1
Cell Culture Media and Reagents
2
SARS-CoV-2 Protein Thermal Stability and Suramin Binding
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SARS-CoV-2 N, NTD and CTD thermal unfolding profiles were acquired by measuring temperature-dependent shift in their intrinsic fluorescence at 330 nm (F330) and 350 nm (F350) emission wavelengths by using a Prometheus NT.48 (NanoTemper Technologies) instrument. Purified proteins were diluted in their storage buffer to 0.3 mg mL−1, 0.13 mg mL−1 and 0.25 mg mL−1, respectively, for the obtainment of optimal fluorescence counts, then loaded on standard capillaries (NanoTemper Technologies) and subjected to a 20–90 °C linear thermal gradient at 0.5 °C min−1 rate. Inflection points of fluorescence transition corresponding to melting temperature (Tm) values were determined as the first derivative maximum of the fluorescence intensity ratio at the measured wavelengths (F330/F350). For interaction between Suramin and the proteins, the Tm shift was assessed after addition to samples of 100 M Suramin (Sigma-Aldrich) water solution and incubation for 1 hour at 25 °C. Suramin affinity to SARS-CoV-2 N NTD and CTD was assessed by titration of the compound over a 0.5–2000 M concentration range against a fixed protein amount. For each experiment, data from at least three independent measurements were processed using the PR. ThermControl (NanoTemper Technologies) software.
3
Evaluation of Antiviral Compounds
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Suramin (Sigma-Aldrich) and 6-azauridine (6-AU) (Sigma-Aldrich) were dissolved in water to produce stock solutions (Suramin, 50 mg/mL, 35 mM; 6-AU, 50 mg/mL, 200 mM) and then stored at −20°C until use. Vero cells were seeded at a density of 1.5 × 104 cells/well (96-well white/clear-bottomed plate). For the Suramin test, the cells were infected with 2,500 IU VRPs/well in the presence of serially diluted Suramin with MEM prior to incubation at 34°C for 1 h. The cells were then grown for another 5 h in 5% FBS medium containing corresponding concentrations of Suramin at 34°C. The 6-AU test involved infecting cells with 2,500 IU VRP/well followed by incubation at 34°C for 1 h. The cells were then grown for another 5 h in 5% FBS medium containing corresponding concentrations of 6-AU at 34°C and then subjected to Luc assay. eGFP (another reporter) expression was assessed by incubating treated cells at 28°C for 20 h. Images were then captured using a Sapphire biomolecular imager (Azure Biosystems).
4
Pharmacological Compound Preparation
5
Overexpression of GRK2 Variants in HaEpi Cells
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The overexpression plasmid construction method: GRK2-GFP-His (ORF of GRK2), GRK2T672A-GFP-His (replacing threonine 672 with alanine) and GRK2S680A-GFP-His (replacing serine 680 with alanine) were overexpressed using the GFP-pIEx-His vector in HaEpi cells. The GPCR inhibitor suramin (sodium salt, final concentration, 50 μM), GDPβs (100 μg/mL) and PKC inhibitor CC (5 μM) (all from Sigma-Aldrich)28 (link) were added to the cells for 60 minutes. Then, HaEpi cells were treated with 20E (1 μM) for 5 minutes. The cells were fixed in 4% paraformaldehyde for 30 min at room temperature. After washing with Dulbecco’s phosphate-buffered saline (DPBS: 8.10 mM Na2HPO4, 1.47 mM KH2PO4, 138 mM NaCl, and 2.67 mM KCl, pH 7.4), the cells were incubated with anti-GRK2 polyclonal antibody (1:500 ratio, diluted in blocking buffer) at 4 °C overnight. The cells were washed with DPBS and then incubated with the secondary antibody goat anti-rabbit-Alexa Fluor 488 (1:1,000 ratio, diluted in blocking buffer) at 37 °C for 1 h. The cells were then stained at the cell membrane using wheat germ agglutinin (WGA, red) for 15 min at room temperature. Pre-serum was used as the negative control. Fluorescence was observed using a Zeiss LSM 700 laser confocal microscope (Zeiss, Thornwood, NY).
6
Enzymatic Osteoarthritis Induction in Mice
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The KU Leuven Ethical Committee for Animal Research approved all animal experiments. Osteoarthritis was enzymatically induced in 8-week-old male wild-type C57/Bl6 mice by intra-articular injection of a mixture of 2% papain (5 µL) (Sigma) and its activator 0.03 M cysteine (5 µL) (Sigma) into the right knee joints on the first day as described before.31–33 (link) Control group received papain only, and the treated group received papain in combination with 0.1 mg suramin (Sigma). An equal volume of sterile PBS was injected into the left knee that served as a control knee. At day 3, mice received a second intra-articular injection of 0.1 mg suramin or PBS. Mice were sacrificed after 6 days, and hind limbs were isolated. These hind limbs were fixed with 4% paraformaldehyde (Millipore) overnight at 4°C. Whole knees were decalcified in 0.5 M EDTA (pH 7.5) for 15 days at 4°C and paraffin embedded to section at 5 µm in frontal plane for histology and TIMP3, VDIPEN and NITEGE immunohistochemistry. Samples were stained with Safranin O and haematoxylin for histology analysis. Articular cartilage damage was quantified by one blinded reader according to the OARSI (Osteoarthritis Research Society International) scoring system.34 (link) Two independent randomised experiments were performed and consisted a total of 30 mice.
7
Identification of Volatile Compounds
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For the identification of volatiles, the following analytical standards purchased from Sigma Aldrich (Milan, Italy) were used: α-pinene, β-pinene, sabinene, 1,8-cineole, camphene, myrcene, α-phellandrene, δ-3-carene, p-cymene, limonene, γ-terpinene, terpinolene, linalool, trans-pinocarveol, terpinen-4-ol, α-terpineol, myrtenal, citronellol, isobornyl acetate, (E)-caryophyllene, α-humulene, (E)-β-ionone, and caryophyllene oxide. The reference drug suramin was purchased from Sigma Aldrich.
8
PolyI:C and Suramin Concentration Evaluation
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9
Probing Plant Ion Homeostasis Mechanisms
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The pharmacological effects of several PM channel inhibitors were tested in salt-treated Arabidopsis seedlings, i.e., amiloride (a PM Na+/H+ antiporter blocker, 50 μM, Sigma-Aldrich, St. Louis, MO, USA) and sodium orthovanadate (a specific inhibitor of the PM H+-ATPase, 500 μM, Sigma-Aldrich, St. Louis, MO, USA) were used to inhibit the PM Na+/H+ antiport system, while tetraethylammonium chloride (TEA, a typical K+ channel inhibitor, 50 μM, Sigma-Aldrich, St. Louis, MO, USA) was used to block the salt-induced K+ efflux [6 (link),7 (link),10 (link)].
The effects of specific antagonists (100 μM PZA, Sigma-Aldrich, St. Louis, MO, USA and 300 μM suramin, Sigma-Aldrich, St. Louis, MO, USA, two inhibitors of the PM ethylene receptor and PM P2-like, respectively) on control and short-term salt-treated Arabidopsis were also evaluated in this study [30 (link),59 (link)]. After the treatment of salt and antagonists, steady-state ion fluxes of the meristematic zone were promptly recorded. Next, the salt-elicited abundances of AtAHA1, AtSOS1, and some salt-related genes were examined in young roots with or without antagonist treatments.
The effects of specific antagonists (100 μM PZA, Sigma-Aldrich, St. Louis, MO, USA and 300 μM suramin, Sigma-Aldrich, St. Louis, MO, USA, two inhibitors of the PM ethylene receptor and PM P2-like, respectively) on control and short-term salt-treated Arabidopsis were also evaluated in this study [30 (link),59 (link)]. After the treatment of salt and antagonists, steady-state ion fluxes of the meristematic zone were promptly recorded. Next, the salt-elicited abundances of AtAHA1, AtSOS1, and some salt-related genes were examined in young roots with or without antagonist treatments.
10
Cisplatin and Suramin Administration
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Cisplatin was obtained as a vial of 10 mg/10 ml saline (Unistin; EIMC United Pharmaceuticals, Badr city, Cairo, ARE). Suramin was purchased from Sigma Aldrich (St. Louis, MO), each 100 mg was dissolved in 10 ml saline (10 mg/ml). All other chemicals used in the experiment were of analytical grade. Suramin was administered by tail vein injection while cisplatin intraperitoneally.
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