The miR-122-5p inhibitor with its antisense or nonsense control lentiviral plasmid was constructed using a synthetic oligonucleotide containing miR-122-5p binding sites or nonsense sequence. The L02 cells were co-transfected with corresponding lentiviral vector constructs and lentiviral mix (miR-122-5p inhibitor and control lentivirus, Thermo Scientific). After 72 h of transfection, the medium was collected and concentrated using Polyethylene glycol 6000 (Sigma) precipitation. The titer was determined using the frequency of the GFP-positive L02 cells.
Polyethylene glycol 6000
Manufactured by Merck Group
Sourced in United States, Germany, India
Polyethylene glycol 6000 is a polymer compound used as a laboratory reagent. It is a water-soluble, non-toxic, and non-reactive material commonly employed in various scientific applications.
Automatically generated - may contain errors
Lab products found in correlation
Sodium chloride (nacl), by Merck Group (3 mentions)
Methanol, by Merck Group (3 mentions)
Enzymatic kit, by Roche (2 mentions)
Tween 20, by Merck Group (2 mentions)
Iron 3 chloride hexahydrate, by Merck Group (2 mentions)
Hydrochloric acid (hcl), by Merck Group (2 mentions)
Acetic acid, by Merck Group (2 mentions)
N n dimethylformamide (dmf), by Merck Group (2 mentions)
Sodium hydroxide (naoh), by Merck Group (2 mentions)
Magnesium stearate, by Merck Group (2 mentions)
Uranyl acetate, by Agar Scientific (2 mentions)
Jem 1011, by JEOL (2 mentions)
Yf 17d 204 stamaril, by Sanofi (1 mentions)
Continuous density gradient fractionator, by Teledyne (1 mentions)
Tobacco acid pyrophosphatase, by Illumina (1 mentions)
T4 rna ligase, by Thermo Fisher Scientific (1 mentions)
Heraeus multifuge x3r, by Thermo Fisher Scientific (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
4 1 1 3 3 tetramethylbutyl phenyl polyethylene glycol triton x100, by Merck Group (1 mentions)
Dioctyl sulfosuccinate, by Merck Group (1 mentions)
39 protocols using polyethylene glycol 6000
1
Lentiviral Inhibition of miR-122-5p
2
Preparation of PEG Solutions
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PEG stocks were prepared by dissolving polyethylene glycol 6000 (Sigma Aldrich, 81,260) in the previously prepared 10 mM citrate 10 mM phosphate buffer to reach a concentration of 15%, 30% and 50% PEG (weight/volume). To dissolve PEG, heating up the solution to around 50 °C is necessary. The pH shifted during the dissolving of PEG and had to be adjusted again. Adjusting the pH of highly concentrated PEG solutions is difficult and takes a considerable amount of time. It is important to let the solution stir for at least 2 min every time acid or base is added to ensure complete mixing before measuring the pH again. Due to this complication, it is suggested to prepare the PEG solutions at least one day in advance of starting the assay. This ensures complete mixing and on the day of the assay the pH can be checked and adjusted again if necessary.
3
Preparation and Titration of YFV Strains
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The YFV-17D vaccine strain (YF-17D-204 STAMARIL, Sanofi Pasteur, Lyon) was provided by the Institut Pasteur Medical Center. The YFV-DAK strain (YFV-Dakar HD 1279) was provided by the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA), through the University of Texas Medical Branch at Galveston, USA. Viral stocks were prepared on Vero cells, concentrated by polyethylene glycol 6000 (Sigma) precipitation and titrated on Vero cells by plaque assay as described previously [22 (link)].
4
Purification and Concentration of FMDV
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A clarified FMDV sample was treated with 7.5% polyethylene glycol 6000 (Sigma-Aldrich) to concentrate the virus particles [38 (link)]. The concentrated viral antigen was placed in a tube with a sucrose density gradient (SDG) of 15–45% and then ultracentrifuged at 100,000× g for 4 h at 4 °C. Centrifuged samples were fractionated using a continuous density gradient fractionator (Teledyne ISCO, Lincoln, Nebraska). The absorbance of each fraction was measured at 254 nm using a spectrophotometer component. At this time, the amount of 146S, a viral particle (μg/mL), was calculated by measuring the area under the peak of a specific fraction according to a previous study [39 (link)]. The peak fraction containing 146S particles was then collected and ultracentrifuged at 100,000× g for 4 h at 4 °C. The harvested virus pellet was resuspended in Tris-KCl buffer (pH 7.6).
5
Viral RNA Extraction and Sequencing
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The clarified supernatant medium of EPEV-infected C6/36 cells was incubated at 4°C overnight in a polyethylene glycol 6000 (Sigma-Aldrich) solution, to concentrate the virions. The concentrate was centrifuged at 3,000 rpm for 30 min, and the pelleted sediments were resuspended and ultracentrifuged at 30,000 rpm for 3 h using a Fiberlite F14-6 × 250y fixed-angle rotor with a Thermo Scientific Heraeus Multifuge X3R ultracentrifuge. The phase 4 fraction of the centrifuge tube was collected to provide the sample that was then used for viral RNA extraction. A 400-μl volume was used for viral RNA extraction as mentioned above and treated with 10 U/μl tobacco acid pyrophosphatase (Epicentre); circularization was performed with 5 U/μl T4 RNA ligase (Ambion) at 10°C overnight. Specific primers were designed to perform RT-PCR and to amplify the extremities using an Access RT-PCR one-step kit (Promega). The positive samples were gel purified using a QIAquick gel extraction kit (Qiagen), and both strands were sequenced.
6
Immobilization of Burkholderia cepacia Lipase
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Carbon nanofiber (CNF), Burkholderia cepacia lipase (BCL), nitric acid, sulphuric acid, vinyl acetate, toluene, surfactants viz. [4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol (Triton X-100), polyoxyethylene (20) sorbitan monooleate, n-octyl-β-d -glucoside (OG), polyethylene glycol 6000 (PEG6000), sodium dodecyl sulphate (SDS), dioctyl sulfosuccinate (DOSS), cetyl trimethyl ammonium bromide (CTAB), merpol], p-nitro-phenyl palmitate (p-NPP), 2-ethanesulfonic acid (MES), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were obtained from Sigma-Aldrich Corporation (St. Louis, MO, USA). HPLC grade solvents were obtained from Merck Ltd.
7
Antithrombin Activity Assay
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Human antithrombin (ATIII)
(R1), purified human thrombin (R2), and chromogenic substrate specific
for thrombin (CS-01(38)) (R3) were purchased from Hyphen-Biomed (France).
European Pharmacopoeia reference standard was purchased from EP (Strasbourg
Cedex, France). Tris(hydroxymethyl)aminomethane, polyethylene glycol
6000, hydrochloric acid, and acetic acid were purchased from Sigma-Aldrich
(USA). Ethylenediaminetetraacetic acid (EDTA) and sodium chloride
(NaCl) were purchased from Merck (Germany). All materials detailed
above were of analytical grade.
(R1), purified human thrombin (R2), and chromogenic substrate specific
for thrombin (CS-01(38)) (R3) were purchased from Hyphen-Biomed (France).
European Pharmacopoeia reference standard was purchased from EP (Strasbourg
Cedex, France). Tris(hydroxymethyl)aminomethane, polyethylene glycol
6000, hydrochloric acid, and acetic acid were purchased from Sigma-Aldrich
(USA). Ethylenediaminetetraacetic acid (EDTA) and sodium chloride
(NaCl) were purchased from Merck (Germany). All materials detailed
above were of analytical grade.
8
Amyloid Protein Labeling Protocol
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Polyethylene glycol 6000 (PEG-6000, catalog number: 528877), Thioflavin-T (ThT, catalog number: 596200), DMSO (catalog number: D8418), and curcumin (catalog number: C1386) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ni-NTA-agarose (catalog number: 30250) was purchased from Qiagen. Cy5.5-curcumin (catalog number: R-CUR-5) was purchased from Ruixibio (Xi’an, China). All other reagents were of analytical grade.
9
Fasting Mice Blood Lipid Analysis
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Every 4 weeks (experiment 1 and 2), mice were fasted for 4 h (9:00 AM to 1:00 PM clock time, corresponding to Zeitgeber time [ZT] 2–6), and subsequently, blood was drawn from the tail vein in capillaries coated with paraoxon to prevent ongoing lipolysis. Blood plasma obtained after centrifugation was used to determine plasma TG and TC levels using enzymatic kits (10166588130 and 11489232216, respectively; Roche Diagnostics). Plasma HDL-C levels were determined in the supernatant after precipitation of ApoB-containing lipoproteins from plasma by addition of 20% polyethylene glycol 6000 (Sigma-Aldrich) in 200 mM glycine buffer (pH 10). From the TC and HDL-C levels, the non-HDL-C levels were calculated.
10
Bacterial Transformation with IPTG/X-Gal
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Medium: 2YT (16 g/L bactotryptone, 1 g/L yeast extract, 5 g/L NaCl, pH 7.0), 1.5% agar and 0.7% agar based on 2YT. Solutions: 1000 × IPTG/X-gal (1.25 g IPTG (isopropyl-β-d -thiogalactoside) and 1 g X-gal (5-bromo-4-chloro-3-indolyl-β-d -galactoside, Sigma, USA) in 25 mL DMF (dimethyl formamide, Sigma, USA), tetracycline 20 mg/mL (Sigma, USA) in 50% ethanol, phosphate buffered saline (PBS), PBST—PBS containing 0.1% Tween-20 (Sigma, USA), blocking solution of 1% gelatin (Sigma, USA) based on PBST, PEG/NaCl precipitating solution (20% (w/v) polyethylene glycol-6000, Sigma, USA, 2.5 M NaCl). Protein markers (Abcam, GB) and DNA electrophoresis markers (Fermentas, Lithuania), conjugate of streptavidin with horseradish peroxidase (Thermo Fisher Scientific, USA).
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