Mice were euthanatized by intraperitoneal injection of pentobarbital sodium (2.5-5 mg per mouse). Lung tissues were obtained 4, 8, or 24 h after challenge, homogenized immediately (Polytron PT2100, Kinematica AG, Littau, Switzerland), and then total RNA was purified from the homogenate with Trizol reagent (Invitrogen Life Technologies Japan, Tokyo, Japan). Total RNA was converted to cDNA using a commercial kit (Quantitect reverse transcription kit, Qiagen, Hilden, Germany) as described previously [25] . Real-time quantitative PCR (qPCR) with intercalation (SYBR green master mix, Qiagen) of the mouse IL-5, IL-13, IL-17, IL-38, and β-actin genes was performed using a thermal cycler (Mx3000p PCR machine, Stratagene, La Jolla, CA). Each specific primer set was purchased from Qiagen Co. (Hilden, Germany). Expression of the mouse IL-5, IL-13, IL-17A and IL-38 genes relative to that of the β-actin gene was calculated using the comparative threshold cycle method, as described previously [13, 25] .
Polytron pt 2100
Manufactured by Kinematica
Sourced in Switzerland, Germany, United States
The Polytron PT 2100 is a high-performance homogenizer designed for a wide range of laboratory applications. It features a powerful motor and a robust construction, making it suitable for efficiently homogenizing, dispersing, and emulsifying a variety of samples. The Polytron PT 2100 is a reliable and versatile piece of equipment for various research and development tasks.
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Lab products found in correlation
Bicinchoninic acid assay, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Sybr green master mix, by Qiagen (1 mentions)
Mastersizer x, by Malvern Panalytical (1 mentions)
Riboerase kit, by Roche (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Nextseq 500 platform, by Illumina (1 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Nuvia imac ni charged resin, by Bio-Rad (1 mentions)
Vivaspin 20 centrifugal concentrator, by Sartorius (1 mentions)
Mini spray dryer b 290, by Büchi (1 mentions)
Lb medium, by BD (1 mentions)
Zorbax hilic plus, by Agilent Technologies (1 mentions)
Exactive orbitrap analyzer, by Thermo Fisher Scientific (1 mentions)
Wx vortex, by VELP Scientifica (1 mentions)
Hesi 2, by Thermo Fisher Scientific (1 mentions)
Alphatrak glucometer, by Abbott (1 mentions)
Trizol, by Kinematica (1 mentions)
30 protocols using polytron pt 2100
1
Mouse Lung Gene Expression Analysis
2
Optimized PWO-in-water Emulsion Design
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Statistical design. PWO-in-water emulsion was performed according to a Box-Behnken experimental design (15 runs, 12 experimental points and three central points). SL content (2–6% of PWO), homogenization time (HT, 1–5 min) and homogenization speed (HS, 11,000–20,000 rpm) were the independent variables. Droplet size (D[4,3]), and lipid oxidation evaluated as thiobarbituric acid reactive substances (TBARs) were the dependent variables.
Experimental data were fit to a second-order regression model. All the experiments were conducted randomly to avoid systematic bias. Response surface methodology (RSM) was applied at each independent variable and the multiple response optimization was performed using the desirability function, where droplet size and TBARs values were minimized.
Preparation and characterization of PWO emulsion. PWO-in-water emulsions (6 g) were prepared by dispersing SL (0.05–0.16 g) in distilled water (3.17–3.28 g) at 40 °C by stirring at 500 rpm for 20 min. The dispersion was added to PWO (2.67 g) and the mixture was homogenized with a Polytron PT-2100 (Kinematica AG, Luzern, Switzerland). Droplet size of PWO emulsions was determined by laser diffraction, using a particle size analyzer (Mastersizer X, Malvern Instruments, Malvern, UK), and expressed as D[4,3]. Lipid oxidation was evaluated by TBARs, according to [20 (link)].
Experimental data were fit to a second-order regression model. All the experiments were conducted randomly to avoid systematic bias. Response surface methodology (RSM) was applied at each independent variable and the multiple response optimization was performed using the desirability function, where droplet size and TBARs values were minimized.
Preparation and characterization of PWO emulsion. PWO-in-water emulsions (6 g) were prepared by dispersing SL (0.05–0.16 g) in distilled water (3.17–3.28 g) at 40 °C by stirring at 500 rpm for 20 min. The dispersion was added to PWO (2.67 g) and the mixture was homogenized with a Polytron PT-2100 (Kinematica AG, Luzern, Switzerland). Droplet size of PWO emulsions was determined by laser diffraction, using a particle size analyzer (Mastersizer X, Malvern Instruments, Malvern, UK), and expressed as D[4,3]. Lipid oxidation was evaluated by TBARs, according to [20 (link)].
3
Liver Microsomal and Cytosolic Fractionation
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Livers were harvested, excised, and snap frozen in liquid nitrogen for storage at −80°C until processing. Briefly, a small piece (∼200 µg) in three volumes of KCl-phosphate buffer (10 mM KHPO4, 20 mM EDTA, and 150 mM KCl, pH 7.4) was homogenized (POLYTRON PT 2100; Kinematica, Lucerne, Switzerland). The homogenate was centrifuged for 80 minutes at 100,000g (30,000 rpm in an F50L rotor) at 4°C. After removal of the fatty layer, the resultant supernatant was centrifuged for 1 hour at 100,000g (30,000 rpm in an F50L rotor; Thermo Scientific, Perth, UK). After ultracentrifugation, the supernatant (cytosolic fraction) was retained and the pellet (microsomal fraction) was resuspended in KCl buffer containing 0.25 M sucrose. The protein content of the microsomal and cytosolic fractions was quantified by bicinchoninic acid assay (Thermo Scientific).
4
RNA Extraction and Sequencing from Myometrium and Leiomyoma
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Thirty milligram of myometrium and leiomyoma tissue was cryopulverized into a fine powder and homogenized in Qiazol lysis reagent using a Kinematica Polytron PT2100 homogenizer. Total RNA was isolated using Qiagen miRNeasy Mini Kit according to the manufacturer’s instructions (Qiagen, cat. # 217004). After RNA elution, an in-solution DNase treatment was performed to completely digest genomic DNA (Qiagen, cat. # 79254). RNA quality was verified using the Bioanalyzer Eukaryote Total RNA 600 Nano Kit (Agilent Technologies, cat. # 5067). Purified RNA samples had a mean RNA integrity number of 8.0. RNA was prepared for sequencing using the KAPA-stranded RNA-seq with RiboErase Kit according to the manufacturer’s instructions (Kapa Biosystems, cat. # KK8483). Paired-end sequencing of all RNA libraries was performed on the Illumina NextSeq 500 Platform.
5
Insulin Signaling in Mouse Tissues
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Mice were fasted for 2 h, given a bolus of insulin (1.5 U/kg) or saline followed by euthanasia by pentobarbital administration followed by decapitation 10 min after bolus. Liver and gastrocnemius muscle were collected and immediately frozen in liquid nitrogen followed by storage at −80°C. Samples were homogenized on ice using a motorized homogenizer (Polytron PT 2100, Kinematica AG) in sodium chloride-Tris-EDTA buffer with 0.1% SDS, 0.1% Triton-X, and 1% HALT protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Cat. No. 78447). The homogenate was sonicated on ice and centrifuged at 16,000 RCF in a cooled centrifuge at 4°C for 20 min, and the supernatant was collected. An aliquot of each sample was diluted and used to quantify the protein concentration using a Lowry assay. The rest of the sample was aliquoted and frozen in liquid nitrogen and stored at −80°C. Protein samples were diluted to 3 µg/µL concentration in STE buffer with 1% HALT protease and phosphatase inhibitor cocktail for Akt (protein kinase B) immunoblot analyses using primary antibodies total (pan) Akt (Cell Signaling, Cat. No. 4691) and phosphorylated Akt (p-Akt) Ser473 (Cell Signaling, Cat. No. 4060) and anti-rabbit IgG HRP-linked secondary antibody (Cell Signaling, Cat. No. 7074) to assess for changes in insulin signaling.
6
Purification of His-tagged Recombinant Plant Proteins
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His-tagged recombinant mZP3-1, GFP-mZP3-1, and mZP3-3 were purified using immobilized metal affinity chromatography (IMAC) based on the affinity for the 6His purification tag. For large-scale protein extraction, 30 g of ground leaf material was homogenized in ice-cooled protein extraction buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM sucrose, 5 mM imidazole) using a Homogenizer (Polytron® PT 2100, Kinematica AG, Malters, Switzerland) for 30 s at 30,000 rpm. Lysates were centrifuged at 4600× g for 15 min and then clarified by repeated ultracentrifugation at 16,000× g for 30 min at 4 °C. The supernatants were collected and loaded onto a column (Bio-Rad Laboratories, Hercules, CA, USA) containing pre-equilibrated Nuvia™ IMAC Ni-charged resin (Bio-Rad Laboratories, Hercules, CA, USA). The column was washed with washing buffer (50 mM NaH2PO4, 20 mM imidazole, 20 mM NaCl, pH 8). The target recombinant protein was further eluted with elution buffer (50 mM NaH2PO4, 300 mM imidazole, 300 mM NaCl, pH 8). The elution fraction was concentrated and desalted using Vivaspin 20 centrifugal concentrators with a 10 kDa cut-off membrane (Sartorius AG, Göttingen, Germany).
7
Emulsion Preparation and Characterization
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Emulsions were prepared by dispersing 0.24 g of powder in 12 mL of distilled water and 12 mL of sunflower oil, following the protocols of Brishti et al. (2017) and Yasumatsu et al. (1972), with adaptations [34 ,36 (link)]. First, samples were mixed at room temperature inside a 50 mL graduated centrifuge tube using a Polytron PT 2100 homogenizer (Kinematica AG, Malters, Switzerland) equipped with a ⌀ 12 mm probe at a speed of 11,000 rpm for 1 min. Subsequently, for the determination of emulsion activity (EA), the samples were centrifuged for 5 min at 1100× g at 20 °C. EA was calculated by measuring the height of the emulsified layer (H1) and the total height of the liquid (HT) and reported as . For the determination of emulsion stability after heat treatment (ES), samples were first heated in a water bath at 80 °C for 30 min, then cooled in an ice-water bath for 15 min, and finally centrifuged for 5 min at 1100× g at 20 °C. For the calculation of ES, the height of the emulsified layer (H2) was recorded. Emulsion stability was reported as .
8
Quantifying Proteins from Tumor Samples
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Protein quantification and western blot were performed as previously described [29 (link)]. For tumor samples, 400 μL of lysis buffer was added to 0.2 g of tumor, and it was disaggregated using a Polytron PT-2100 (Kinematica AG, Malters, Switzerland). Once the tissue was homogenized, the above-mentioned protocol was followed.
9
LPMO-Assisted Kraft Fiber Pretreatment
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Kraft fibers (100 mg) were adjusted to pH 5.2 with sodium acetate buffer (50 mM) in a final reaction volume of 20 mL with 1 mM l -cysteine. Purified LPMO enzyme was added to the substrate at a final concentration of 1.6 µM. Enzymatic incubation was performed at 50 °C under mild agitation for 16 h. Samples were then dispersed with a Polytron PT 2100 homogenizer (Kinematica AG, Germany) for 3 min then ultrasonicated with a QSonica Q700 sonicator (20 kHz, QSonica LLC, Newtown, CT) at 350 W ultrasound power for 3 min. The reference sample was submitted to the same treatment but did not contain the LPMO enzyme.
10
Beef Lipid Extraction Protocol
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Samples of LM taken between the 12th and 13th ribs were removed from each carcass and transported to the laboratory while kept at 4°C. After trimming the subcutaneous fat, the samples were homogenized and stored in a freezer at −80°C until analysis. Total lipids were extracted from the beef samples using the modified method by Folch et al. (1957) (link). Approximately 5 g of muscle tissue was homogenized (Polytron PT2100, Kinematica Inc., Bohemia, NY, USA) with 5 mL of chloroform:methanol (2:1, vol:vol) solution and incubated at room temperature for 30 min. The homogenate was filtered through Whatman GF/C filters (Whatman Ltd., Maidstone, UK) and rinsed with an additional 10 mL of chloroform:methanol solution. The extracted lipid was combined with 8 mL of 0.74% KCl and stirred for 1 min. After the phases separated, the lipid layer was transferred to 20-mL scintillation vials (Wheaton, Millville, NJ, USA) and the solvents were evaporated by heating (C-WBE, Changshin Science, Seoul, Korea) at 60°C under nitrogen.
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