Grape marc from red grape (Carménère variety) was supplied by Concha y Toro winemaker (Pencahue, Chile) and stored at −80 °C until use. An ultrasound-assisted extraction (UAE) was carried out in an ultrasonic system (SFX550 Sonifier, Branson Ultrasonics Corporation, Danbury, CT, USA) in optimal operating conditions: grape marc, 23.85% w/w; ethanol, 40% w/w; water, 36.15% w/w; amplitude, 20%; temperature, 22 °C; operating time, 15 min. The experimental set-up for the extraction process was illustrated in our previous work [5 ]. The extracted solution was filtered on nylon cloth and stored at −5 °C until its clarification by microfiltration (MF). The clarification was carried out using a mono-tubular ceramic membrane (Tami Industries, Nyons, France) with a pore size of 0.14 μm and an effective membrane area of 0.005 m2. The original hydraulic permeability of this membrane was 0.57 L/m2bar. The clarification process was operated at a transmembrane pressure (TMP) of 2.25 bar, a temperature of 25 °C and an axial feed flow rate (Qf) of 4.93 L/min according to the batch concentration configuration (recycling the retentate stream in the feed reservoir and collecting the permeate stream separately).
Sonifier sfx550
Manufactured by Emerson
Sourced in United States
The Sonifier SFX550 is a laboratory-grade ultrasonic processor designed for sample preparation and cell disruption applications. It utilizes high-frequency sound waves to generate cavitation, which can be used to break down materials and homogenize samples.
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Lab products found in correlation
Edta free protease inhibitor, by Roche (1 mentions)
His probe, by Santa Cruz Biotechnology (1 mentions)
Bca reagent, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Protein g agarose, by Merck Group (1 mentions)
Mnase, by Takara Bio (1 mentions)
Protein g dynabead, by Thermo Fisher Scientific (1 mentions)
Anti p300, by Active Motif (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Precellys homogenizer, by Bertin Technologies (1 mentions)
14 protocols using sonifier sfx550
1
Ultrasound-Assisted Extraction of Grape Marc
2
Grape Marc Ultrasound-Assisted Extraction
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Grape marc from red grape (Carménère variety) was supplied by Concha y Toro winemaker (Pencahue, Chile) and stored at −80 °C until use. The ultrasound-assisted extraction (UAE) was carried out in an ultrasonic bath system (SFX550 Sonifier, Branson Ultrasonics Corporation, Danbury, CT, USA) in optimal operating conditions: grape marc, 23.85% w/w; ethanol, 40% w/w; water, 36.15% w/w; amplitude, 20%; temperature, 22 °C; operating time, 15 min. The experimental setup for the extraction process is illustrated in Figure 1 . The total extracted solution (10 L), in sequential steps, was filtered on nylon cloth and stored at −5 °C until its characterization and clarification by MF.
3
Antimicrobial Fusion Protein Expression in E. coli
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Escherichia coli BL21/pET22b cultures (0.5 L) with each antimicrobial fusion (LAP-GFP-H6, and HD5-GFP-H6) were grown overnight (O/N) in shake flasks at 37 °C and 250 rpm in LB broth with ampicillin at 100 μg/mL. Escherichia coli Origami B/pET22b with each antimicrobial fusion (LAP-GFP-H6 and HD5-GFP-H6) were grown at the same conditions with ampicillin, kanamycin, and tetracycline at 100, 25, and 12.5 μg/mL, respectively. The O/N were used as inoculum in fresh LB medium, starting at OD600 = 0.05. Recombinant protein expression was induced by 1 mM IPTG when cultures reached an OD600 = 0.4–0.6. Culture samples of 25 mL were withdrawn at 0, 1, 3, and 5 h post-induction, and they were collected by centrifugation at 6000×g for 15 min at 4 °C. Pellets were resuspended in 500 μL PBS with EDTA-free protease inhibitor (Roche) and bacteria were disrupted by sonication (2 cycles of 3 min, 0.5 s on, 0.5 s off at 10% amplitude) (Branson SFX550 Sonifier). Soluble and insoluble fractions were split by centrifugation (15,000×g, 15 min, 4 °C). Quantifications of LAP-GFP-H6 and HD5-GFP-H6 in both BL21 and Origami strains were obtained by western blot using a monoclonal anti-His antibody (His-probe, Santa Cruz), and their purity was evaluated by a Coomassie blue staining assay. Both outcomes were evaluated by ImageJ software to determine protein quantity and purity.
4
Sonication of Mango Juice at High Temperature
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Sonication was performed on 150 mL mango juice in a 250 mL glass bottle. The ultrasonic processor of 550 W (Branson SFX550 Sonifier, Nuevo Laredo, Mexico), with a 0.5-inch probe operating at 20 kHz frequency was used for sonication. Samples were treated at 60 °C for thermosensation and less than 25 °C for sonication by radiating 100% of power (550 W) for 8 min and, keeping pulse durations of 5 s. Overheating of the samples was prevented by circulating ice water through the treatment chamber. Thermosonication (TS) of samples was started when the set temperature at 60 °C. The observed rise in temperature of the samples due to sonication was 2–5 °C.
5
Vesicular Stomatitis Virus Infection Kinetics
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HeLa cells grown to confluency (90–95%) were either mock-infected or infected with wt-VSV at a Multiplicity of Infection (MOI) of 10 for 1 h. The cells were washed once with PBS, and the media were replaced with DMEM containing 2% FBS and 1XPSG. The cells (attached as well as floating) from the VSV-infected and mock-infected dishes were lysed with 1% SDS at specific time points from 0 to 24 h. The lysates were boiled at 100 °C for 5 min, and then sonicated at an amplitude of 65% for 5 min (Branson SFX550 Sonifier, Danbury, CT, USA). The protein concentration of the lysates was then estimated using a BCA reagent (Thermo-Pierce, Chicago, IL, USA), as per the manufacturer’s protocol. In total, 30 μg cell lysate protein was separated on a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane. The blots were probed with specific primary antibodies against human CD46, CD55, VSV M protein, and human β-actin, and their respective HRP-conjugated secondary antibodies. The blots were developed using SuperSignal West Pico chemiluminescent substrate (Thermo-Pierce, Chicago, IL, USA), and were exposed to X-ray film. The blots were stripped in 0.2 M glycine HCl pH 2.3 and re-probed. Three independent infection experiments and Western blots were performed in order to verify the observation.
6
Silk Microparticle-based PUF Tags
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Each of the four silk MPs was first suspended in water to a concentration of 1 wt %, followed by homogenization of the suspension through tip sonication (Branson SFX 550 Sonifier). Each silk MP suspension was sonicated in an ice bath for 5 min with an amplitude of 30% (165-W output power) and pulse durations of 10 s on time and 10 s off time. Following sonication, the 1 wt % suspensions of four silk MPs were well mixed at a volume ratio of 2:1:1:1 by pipetting up and down. Regenerated silk fibroin solution at 1 wt % was then added to the mixed MP suspension at a volume ratio of 3:7, generating a final suspension made of 0.7 wt % (i.e., 7 mg/ml) silk MPs and 0.3 wt % (i.e., 3 mg/ml) amorphous silk molecules. To fabricate spectral PUF tags on seeds, 5 μl of the final suspension was drop cast on each seed and air-dried.
For visual PUF tags, 100 mM FeCl3 solution was added to the above silk MPs and amorphous silk suspension at a volume ratio of 1:1, generating a final suspension made of 50 mM FeCl3, 0.35 wt % silk MPs, and 0.15 wt % amorphous silk. To fabricate visual PUF tags on seeds, 10 μl of the final suspension was drop cast on each seed and air-dried.
For visual PUF tags, 100 mM FeCl3 solution was added to the above silk MPs and amorphous silk suspension at a volume ratio of 1:1, generating a final suspension made of 50 mM FeCl3, 0.35 wt % silk MPs, and 0.15 wt % amorphous silk. To fabricate visual PUF tags on seeds, 10 μl of the final suspension was drop cast on each seed and air-dried.
7
Quantification of Cellular Triglycerides
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After treatment, the medium was removed, and cell extracts were used for triglyceride determination. AML12 cells were washed extensively with phosphate-buffered saline (PBS), and the suspension was sonicated in 10 mM Tris-HCl pH 7.4, 150 mM NaCl and 1 mM EDTA on ice with five 5-s bursts in a Branson Sonifier SFX550 (San Luis, Misuri, MO, USA) fitted with a microtip. Furthermore, triglyceride content was measured with a commercial kit (Spinreact, Girona, Spain). Protein measurements were performed using the Bradford method [26 (link)]. Triglyceride content values were obtained as mg triglycerides/mg protein and expressed as the percentage of the control cells.
8
Triglyceride Quantification in Emulsions
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The triglyceride (TAG) content in the obtained emulsions was measured using a colorimetric method52 (link),53 (link). In brief, emulsion samples were diluted with 2 wt.% Tween 20 to a range of 0.5–4 g oil/L. Next, the droplets were broken up in smaller droplets by sonication with the Branson Sonifier SFX550 (Brookfield, CT, USA), equipped with a 1/8 inch tapered microtip (Branson, Brookfield, CT, USA), at an amplitude of 35% for 15 s. This was done to increase the oil–water interface for effective hydrolysis of triglycerides and thereby to obtain accurate results. The droplet size distribution of the broken droplets was independent of the initial droplet size, as shown previously36 (link). Next, about 20 µL of sample were weighed into a 2-mL microtube, and 1 mL of assay reagent was added. The samples were incubated in a temperature-controlled block, in the dark, and shaken at 850 rpm, at 20 °C, for 20 min. The absorbance was measured at a wavelength of 500 nm. The TAG content was determined using a calibration curve of a standard TAG dispersion (0.5–4 g oil/L).
9
Crosslink and Native ChIP-seq protocol
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Crosslink ChIP: WT-, GFP- and GN-DU145 cells (2 × 107) were fixed with 1% formaldehyde (Sigma) and sonicated using the Sonifier SFX550 instrument (BRANSON, CT, USA) to induce chromatin fragmentation. Anti-NANOG (CST; #5232) and anti-p300 (Active Motif, CA, USA; #61401) Abs were bound to Protein G Dynabeads (Thermo Fisher Scientific, MA, USA), and these conjugates were used for ChIP. Native ChIP: Nuclear fractions of WT-, GFP- and GN-DU145 cells (1 × 106) were treated with 1200 U MNase (Takara, Shiga, Japan) for chromatin fragmentation. The anti-H3K27Ac Ab (Abcam, Cambridge, UK; ab4729) was bound to Protein G-agarose (Merck Millipore), and the conjugate was used for ChIP. The ChIPseq library was prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) and the NEBNext Multiplex Oligos for Illumina. ChIPseq libraries were sequenced by Illumina.
10
Chitin Nanocrystal Preparation by Acid Hydrolysis
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A general acid hydrolysis procedure was used to prepare chitin nanocrystals, as described in [16 (link),20 ]; the effects of production conditions on nanocrystal properties are extensively reviewed in [18 (link)]. Crude chitin powder was hydrolyzed in 3 M HCl for 90 min at 90 °C, after which the reaction was stopped by cooling the mixture on ice. To remove the HCl, the mixture was centrifuged at 4000× g for 5 min, after which the supernatant was discarded, and the pellet redispersed in water. The latter steps were repeated three times. This suspension was sonicated with the Branson Sonifier SFX550 (Brookfield, CT, USA) equipped with a sonication tip 1/8′ tapered microtip (Branson, Brookfield, CT, USA), in pulses of 100 J at an amplitude of 40% with 10 s rest, and a total sonication energy of ~150 J/mL while cooled on ice. To collect the chitin nanocrystals (ChNC), two centrifugation steps were applied (1000× g, 15 min), after which the pellet was discarded.
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