AKTA purifier 100 protein separation and purification system (GE Healthcare Co., USA). Beacon 2000 fluorescence polarization analyzer (PanVera LLC, USA). Synergy 4 microplate reader (BioTek Instruments, Inc., USA). SHIMADZU UV-3600 ultraviolet and visible spectrophotometer (Shimadzu Corp., Japan). VCX-500 ultrasonic processor (Sonics & Materials, Inc., USA). Inverted microscope (Nikon Corp., Japan). Gradient thermal cycler (Eppendorf Co., Germany). Milli-Q ultra pure water system (Millipore Corp., USA).
Vcx 500 ultrasonic processor
Manufactured by Sonics
Sourced in United States
The VCX-500 Ultrasonic Processor is a laboratory equipment device that utilizes high-frequency sound waves to process and disrupt samples. It is designed to provide controlled and efficient sample preparation for a variety of applications.
Automatically generated - may contain errors
Lab products found in correlation
Versamax microplate reader, by Molecular Devices (2 mentions)
Centrifuge 5424r, by Eppendorf (2 mentions)
Gradient thermal cycler, by Eppendorf (1 mentions)
Milli q ultrapure water system, by Merck Group (1 mentions)
Synergy 4 microplate reader, by Agilent Technologies (1 mentions)
Inverted microscope, by Nikon (1 mentions)
Optima xl 100k ultracentrifuge, by Beckman Coulter (1 mentions)
Pierce bicinchoninic acid bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Lightcycler 2.0 instrument, by Roche (1 mentions)
Protein a agarose beads, by Merck Group (1 mentions)
Dneasy plant mini kit, by Qiagen (1 mentions)
Fecl2 4h2o, by Merck Group (1 mentions)
Fecl3 6h2o, by Merck Group (1 mentions)
Calcium detection assay kit, by Abcam (1 mentions)
Microplate reader, by Molecular Devices (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
8 protocols using vcx 500 ultrasonic processor
1
Protein Separation and Purification System
2
CYP2E1 Activity Assay in Liver Microsomes
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Liver tissues (500 mg) homogenized in 5 mL of 0.15 M KCl were sonicated for 10 s in an ice bath with a VCX-500 Ultrasonic Processor (Sonics and Materials Inc., Newton, CT, USA). Tissue lysate was subjected to centrifugation at 9000× g for 15 min, and the supernatant was further centrifuged at 105,000× g for 1 h using the Optima XL-100K Ultracentrifuge (Beckman, Palo Alto, CA, USA). The microsomal pellet was collected and resuspended in 200 µL of 0.15 M KCl. Protein concentration in microsomal lysates was quantified using a PierceTM bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific). Microsomal protein (100 µg) was incubated in 100 µL reaction buffer (0.1 M KH2PO4/K2HPO4, pH 7.4, 0.4 mM p-nitrophenol, 1 mM NADPH) at 37 °C in a shaking water bath for 1 h. At the end of incubation, 30 µL of 20% trichloroacetic acid (TCA) was added to stop the reaction, and the mixture was subjected to centrifugation at 10,000× g for 10 min. The supernatant was collected and mixed with 10 µL of 10 N NaOH. The absorbance of the supernatant was measured at 510 nm using the VERSAmax™ microplate reader (Molecular Devices). CYP2E1 activity (pmol/mg/min) was calculated by multiplying sample absorbance by an extinction coefficient of 9.53 × 105 M−1·cm−1.
3
Chromatin Immunoprecipitation (ChIP) Protocol for Rice Protoplasts
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For ChIP assays, the Ubipro:GFP and Ubipro:OsWRKY5-GFP constructs were transfected into rice protoplasts as previously described (Zhang et al., 2011b (link)). Protoplasts were then subjected to crosslinking with 1% formaldehyde in W5 solution (154-mM NaCl, 125-mM CaCl2, 5-mM KCl, and 2-mM MES [pH 5.8]) for 30 min under vacuum. Nuclei were isolated and lysed, and chromatin complexes were purified and sonicated, as described previously (Saleh et al., 2008 (link)). DNA was sonicated using a VCX500 ultrasonic processor (Sonics, Connecticut, USA). Anti-GFP polyclonal antibody, ChIP grade (ab290; Abcam, Cambridge, UK) and protein A agarose beads (Merck Millipore) were used for immunoprecipitation. DNA recovered from agarose beads was purified using the DNeasy Plant Mini Kit (Qiagen, Weinbunt, Germany). qPCR analysis was conducted on a LightCycler 2.0 instrument (Roche Diagnostics, Basel, Switzerland) using the following conditions: 95°C for 5 s, 59°C for 15 s and 72°C for 10 s. OsGAPDH (Os04g40950) was used as a negative control. Gene-specific primers are listed in Supplemental Table S2 .
4
Graphene-based Polymer Grafting via Sonochemistry
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The overall preparation was based on sonochemistry. Two grafting schemes were explored. Scheme 1 was the radical-free option using non-covalent interactions based on electron sharing (i.e. π–π bonds). We used pyrene-terminated PS (i.e. Pyr-PSa) whose electronic structure is close to that of graphene. Scheme 2 used radicals to covalently graft PS–Co/C. The radicals originate from in situ polymerization of monomers of styrene. Firstly, deagglomeration of Co/C powders is prerequisite. It was performed by ultra-sonication at 100 W during 10 min, using a VCX500 Ultrasonic processor from Sonics. Note that the duration of sonication must not exceed 30 min because graphene coating deteriorates for longer sonication times.
5
Synthesis of Coated SPIO Nanoparticles
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Dextran-, UFH- and LMWH-coated SPIO nanoparticles were synthesized as described in previous reports [18 (link), 19 (link)]. Briefly, SPIOs were synthesized via co-precipitation with FeCl2•4H2O (185 mg; Sigma, St. Louis, MO, USA) and FeCl3•6H2O (500 mg; Sigma). They were dissolved in deoxygenated distilled water (30 ml), and ammonium hydroxide solution (NH4OH, 7.5 ml) was added under nitrogen (N2) gas while stirring for 30 min. After removing unnecessary salt in the solution, 250 mg of either dextran (m.w. 40 kDa; Sigma), UFH, or LMWH (Nanjing King-Friend Biochemical Pharmaceutical Co., Ltd., Nanjing, China) was added to 20 ml of distilled water. Each solution was stirred for 2 h at room temperature and then sonicated (200 W; VCX-500 Ultrasonic Processor; Sonics & Materials, Inc., Newtown, CT, USA) for 60 min. The supernatant was heated at 80 °C for 1 h and applied to a magnetic field for 6 h. Finally, the solutions were centrifuged to remove aggregated particles at 4000 rpm for 10 min and stored at 4 °C until use.
6
Calcium Quantification in Liver Tissue
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The calcium concentration was measured using a Calcium Detection Assay Kit (Abcam) according to the manufacturer's protocol. Liver tissues (100 mg) were homogenized in 500 µl of calcium assay buffer and then sonicated for 10 s in an ice bath with a VCX‐500 Ultrasonic Processor (Sonics and Materials Inc.). The homogenate was centrifuged at 12000 × g for 5 min (Eppendorf Centrifuge 5424R, Eppendorf AG). After centrifugation, the supernatant was transferred to a clean tube and used as the tissue lysate. Calcium standards were prepared in serial dilutions of 0, 0.2, 0.4, 0.6, 0.8, and 1 mM by diluting the 5 mM standard stock in distilled water. The reaction was set up by mixing 50 µl of calcium standard or tissue lysates, 90 µl of chromogenic reagent, and 60 µl of calcium assay buffer into a 96‐well plate, and then incubating for 10 min at room temperature in the dark. The absorbance at 575 nm was measured using a VERSAmax™ microplate reader (Molecular Devices). The calcium concentration (mM) was calculated by dividing the sample amount from the standard curve by the sample volume added into the well and multiplying by the sample dilution factor.
7
Measuring Inflammatory Cytokines in Liver
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The concentrations of the pro‐inflammatory cytokines IL‐1β, IL‐6, and TNF‐α in the liver tissues were quantified using an ELISA kit (R&D system, Minneapolis, MN, USA) according to the manufacturer's protocol. Liver tissues (50 mg) homogenized in 1 ml of 1 × PBS were sonicated for 10 s in an ice bath with a VCX‐500 Ultrasonic Processor (Sonics and Materials Inc.). The homogenates were subjected to centrifugation at 12,000 × g for 5 min (Eppendorf Centrifuge 5424R, Eppendorf AG). After centrifugation, the supernatant was transferred to a clean tube and used as the tissue lysate. Tissue lysates (50 μl) were added to the wells of 96‐well plates, which were precoated with anti‐IL‐1β, IL‐6, and TNF‐α antibodies. The plates were covered with an adhesive strip, incubated for 2 h at room temperature, and washed three times with a wash buffer. Then, 100 μl of mouse IL‐1β, IL‐6, and TNF‐α conjugate was added, and the plates were incubated for 2 h at room temperature, and washed three times. The reaction was quenched by the addition of 100 μl of stop solution. The absorbance of the plates at 450 nm was read with a microplate reader (Molecular Devices).
8
Protein Extraction and Immunoblotting in Endothelial Cells
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Endothelial cells (7.5×105) were washed with ice-cold PBS and scraped off the flask into 100 μl lysis buffer containing 25 mM Tris-HCl, pH 6.8, 1% sodium dodecylsulfate, 1 mM phenylmethylsulphonyl fluoride and protease inhibitor cocktail (Sigma). The resultant lysates were further disrupted by sonication for 10 sec at an amplitude of 35% using a VCX 500 Ultrasonic Processor (Sonics & Materials, Newtown, CT, USA) and then centrifuged at 12,000 × g for 20 min to remove particulate material. Proteins (30 μg) were fractionated by SDS-PAGE and transferred to PVDF membranes. The membranes were incubated as previously described (12 (link)).
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