Manufactured by Vibra-Cell
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The Ultrasonic Processor is a laboratory equipment designed to produce high-frequency vibrations. It is used to disrupt materials, mix solutions, or emulsify liquids through the application of ultrasonic energy.
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9 protocols using Ultrasonic processor
1
Quantification of the protein expression levels of pro-inflammatory cytokines
In order to examine the effects of melatonin on the protein expression levels of specific pro-inflammatory cytokines in the hippocampus of the CCH rat model, enzyme linked immunosorbent assays (ELISA) for tumor necrosis factor (TNF)-α and interleukin (IL)-1β were performed, in accordance with a previous study (26 (link)). Briefly, the rats (n=5/group) were sacrificed 28 days following surgery, and the hippocampi were removed and homogenized using a Vibra-Cell ultrasonic processor. After centrifugation of the homogenates at 14,000 × g for 20 min at 4°C, the supernatant was collected. The levels of TNF-α and IL-1β were examined using a commercial Invitrogen ELISA kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's protocol.
LEE C.H., PARK J.H., AHN J.H, & WON M.H. (2016). Effects of melatonin on cognitive impairment and hippocampal neuronal damage in a rat model of chronic cerebral hypoperfusion. Experimental and Therapeutic Medicine, 11(6), 2240-2246.
2
Norepinephrine Measurements
NE concentration in TG was measured using liquid chromatography (Agilent, USA) as previously described.26 Briefly, mice (n = 6 per group) were anesthetized and transcardially perfused with 30 mL ice-cold PBS at 7 dpi. The TGs were isolated, weighed, and homogenized at 4°C by a Vibra-Cell ultrasonic processor supplemented with 500 μL 0.14% sodium heptane sulfonate solution (pH 3.0 ± 0.1) five times for 10s with intervals of 10s at 60 Hz. The mixture was centrifuged at 4°C, 12,000 rpm for 15 minutes. The supernatant was collected and sonicated before being injected onto the liquid chromatography system employing the fluorescence method. Quantification was achieved by a series of standard solutions.
Mai L., Jia S., Liu Q., Chu Y., Liu J., Yang S., Huang F, & Fan W. (2022). Sympathectomy Ameliorates CFA-Induced Mechanical Allodynia via Modulating Phenotype of Macrophages in Sensory Ganglion in Mice. Journal of Inflammation Research, 15, 6263-6274.
3
Chromatin immunoprecipitation assay
In vitro-differentiated Th cells were activated with anti-CD3 for 3 hours and were crosslinked for 15 min with 1% formaldehyde at RT with rotation. The reaction was quenched by adding 0.125 M glycine and incubated at RT for 5 min. Fixed cells were lysed with cell lysis buffer, followed by nuclear lysis buffer. Nuclei were degraded and chromosomal DNA were fragmented to a size range of 200–500 bp through ultrasonic processor (Vibra-cell). After sonication, the supernatant was diluted 10-fold with ChIP dilution buffer. After pre-clearing, the supernatant was incubated with the ChIP antibodies at 4 °C overnight with rotation. The following day, immunocomplexes were precipitated with Protein Agarose A or G beads at 4 °C for 2–4 h. Immunocomplexes were washed with low salt, high salt, LiCl and two times with TE buffer. After elution followed by reverse crosslinks, DNA was purified and analyzed by qPCR. After normalization to the Input DNA, the amount of output DNA of each target protein was calculated by subtracting that of the IgG control. Quantification of ChIP assay by qPCR was performed using primers as described (38 (link)).
Kharwadkar R., Ulrich B.J., Qayum A.A., Koh B., Licona-Limon P., Flavell R.A, & Kaplan M. (2020). Expression efficiency of multiple Il9 reporter alleles is determined by cell lineage. ImmunoHorizons, 4(5), 282-291.
4
ATP synthase extraction and blue native page analysis
Hela cells were harvested and washed with chilled PBS (pH 7.4) for three times. The harvested cell pellets were re-suspended in hypotonic buffer A (10 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, pH 7.5) with the presence of protease inhibitor and then lysed by Vibra-Cell Ultrasonic Processor with Sound Abating Enclosure. The cell debris and unbroken cells were removed by centrifugation (3000 × g for 10 min at 4 °C). The supernatant was further centrifuged (20,000 × g for 30 min at 4 °C) and mitochondria were collected from the pellet. The mitochondria were lysed by 1% digitonin in ice-water bath for 30 min. The solubilized mitochondria lysate was collected by centrifugation of 30 min at 20,000 × g. The supernatants were treated with benzonase and centrifuged again. The soluble complexes were analyzed by blue native page (BNP) 4 °C in 4–16% acrylamide gradient Bis-Tris gels (Thermo Fisher Scientific) according to the manufacturer’s instructions.
Wang H., Hu L., Li H., Lai Y.T., Wei X., Xu X., Cao Z., Cao H., Wan Q., Chang Y.Y., Xu A., Zhou Q., Jiang G., He M.L, & Sun H. (2023). Mitochondrial ATP synthase as a direct molecular target of chromium(III) to ameliorate hyperglycaemia stress. Nature Communications, 14, 1738.
5
Chromatin immunoprecipitation
In vitro-differentiated Th9 cells or bone marrow derived mast cells were crosslinked for 15 min with 1% formaldehyde at RT with rotation. The reaction was quenched by adding 0.125 M glycine and incubated at RT for 5 min. Cells were lysed with cell lysis buffer, followed by nuclear lysis buffer. Nuclei were degraded and chromosomal DNA were fragmented to a size range of 200–500 bp through ultrasonic processor (Vibra-cell). Post sonication, supernatant was diluted 10-fold with ChIP dilution buffer. After pre-clearing, the supernatant was incubated with the ChIP antibodies at 4 °C overnight with rotation. Immunocomplexes were precipitated with Protein Agarose A or G beads at 4 °C for 2–4 hours. Immunocomplexes were washed with low salt, high salt, LiCl and two times with TE buffer. After elution followed by reverse crosslinks, DNA was purified and analyzed by qPCR. Once normalized to Input DNA, the amount of output DNA of each target protein was calculated by subtracting that of the IgG control. ChIP antibodies are listed in
. Primers used are previously defined (5 (link)).
Qayum A.A., Koh B., Martin R.K., Kenworthy B.T., Kharwadkar R., Fu Y., Wu W., Conrad D.H, & Kaplan M.H. (2019). The Il9 CNS-25 regulatory element controls mast cell and basophil IL-9 production. Journal of immunology (Baltimore, Md. : 1950), 203(5), 1111-1121.
6
Chromatin immunoprecipitation
In vitro-differentiated Th cells were crosslinked for 15 min with 1% formaldehyde at RT with rotation. The reaction was quenched by adding 0.125 M glycine and incubated at RT for 5 min. Fixed cells were lysed with cell lysis buffer, followed by nuclear lysis buffer. Nuclei were degraded and chromosomal DNA were fragmented to a size range of 200–500 bp through ultrasonic processor (Vibra-cell). After sonication, the supernatant was diluted 10 fold with ChIP dilution buffer. After pre-clearing, the supernatant was incubated with the ChIP antibodies at 4 °C overnight with rotation. The following day, immunocomplexes were precipitated with Protein Agarose A or G beads at 4 °C for 2–4 h. Immunocomplexes were washed with low salt, high salt, LiCl and two times with TE buffer. After elution followed by reverse crosslinks, DNA was purified and analyzed by qPCR. After normalization to the Input DNA, the amount of output DNA of each target protein was calculated by subtracting that of the IgG control. ChIP antibodies and primer sequences are listed in Supplementary Table
and
.
Koh B., Abdul Qayum A., Srivastava R., Fu Y., Ulrich B.J., Janga S.C, & Kaplan M.H. (2018). A conserved enhancer regulates Il9 expression in multiple lineages. Nature Communications, 9, 4803.
7
Formulation of Diclofenac prodrugs as nanoparticles (NPs)
Nanoparticles were prepared via an emulsion-evaporation process. DSPE-PEG 2000 is used as excipient to avoid Diclofenac recrystallization and ensure formation of stable nanoparticles with maximal encapsulation of prodrugs (Gaudin et al., 2014; (link)Leng et al., 2014; (link)Lorscheider et al., 2019a; Maksimenko et al., 2014; (link)Quan et al., 2014) (link). The lipophilic prodrug(s) (25 mg) and DSPE-PEG 2000 (12.5 mg) were dissolved in 1 mL chloroform at a 2:1 prodrug to DSPE-PEG 2000 ratio (w:w). The prodrug and DSPE-PEG 2000 mixture was injected into 5 mL of deionized water that pre-chilled (4 °C) and then vortexed for 30 s to form a crude emulsion. Following that, 3 minutes of probe sonication (Sonics, Ultrasonic processor, Vibra-Cell, model: VCX130PB, USA) was conducted. A rotary evaporator with a bath temperature of 60 °C was then used to evaporate the organic solvent, at a pressure of 300 mbar, initially for the first 30 minutes, and then a pressure of 200 mbar for the following 30 minutes. The final suspension volume was then completed to 5 mL and kept at room temperature and 4 °C until further examination.
Hussain S., Ur-Rehman M., Arif A., Cailleau C., Gillet C., Saleem R., Noor H., Naqvi F., Jabeen A., A.t.t.a.-.U.r.-.R.a.h.m.a.n., Iqbal Choudhary M., Fattal E, & Tsapis N. (2023). Diclofenac prodrugs nanoparticles: An alternative and efficient treatment for rheumatoid arthritis?. International journal of pharmaceutics, 643.
8
Determination of protein carbonylation
The content of protein carbonyl products generated by glycoxidation of hepatic cells was measured as previously described 2623 . Briefly, cells were lysed in PBS (pH 7.4) with an ultrasonic Processor (Vibra-Cell, Connecticut, USA). Then, the extracts were centrifuged (10000g, 15 min) and supernatants were collected. Absorbance was measured at 360 nm and carbonyl content was expressed as nmol mg -1 protein using an extinction coefficient of 22000 nmol/L/cm. Protein concentration by using the Bradford reagent.
Navarro M., Morales F.J, & Ramos S. (2017). Olive leaf extract concentrated in hydroxytyrosol attenuates protein carbonylation and the formation of advanced glycation end products in a hepatic cell line (HepG2). Food & function, 8(3).
9
Reagents and equipments
2,2-diphenyl-1-picrylhydrazyl (DPPH), Folin-Ciocalteu reagent, 3,4,5-trihydroxybenzoic acid (Gallic acid) and 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) were purchased from Sigma-Aldrich (Munich, Germany). Ethanol, potassium chloride and sodium carbonate were purchased from Avantor Performance Materials (Gliwice, Poland). Sodium acetate was purchased from Chempur (Piekary Śląskie, Poland). All chemicals and reagents were of analytical grade.
The freeze-drying process was carried out using a TG15 lyophilizer by an external company Naturim (Włocławek, Poland) . The entire extraction process used a Vibra-Cell ultrasonic processor (130 Watt, 20 kHz). Spectrophotometric assays were performed using a V-770 double-beam Jasco spectrophotometer.
Sady S., Matuszak L, & Błaszczyk A. (2019). Optimisation of ultrasonic-assisted extraction of bioactive compounds from chokeberry pomace using response surface methodology. Acta scientiarum polonorum. Technologia alimentaria, 18(3).
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