The levels of total disaccharides of HS in C4-2 cells were determined as described previously69 (link). In brief, cells were collected using a rubber scraper, sonicated using an Ultrasonic homogenizer (Taitec), and treated exhaustively with actinase E (Kaken Pharm). The remaining proteins and peptides were precipitated with trichloroacetic acid, and then extracted with ether to remove trichloroacetic acid. The resultant crude GAG-peptide fractions were desalted by an Amicon Ultra-4 (3 K, Millipore), and treated with heparinase-I, heparinase-III (IBEX Pharmaceuticals), and heparinase-II (R&D Systems) mixture to analyze the disaccharide composition of HS and heparinase-resistant oligosaccharides, which may contain 3-O-sulfated GlcN residues. The digested samples were labeled with a fluorophore 2-aminobenzamide (2AB), and the resulting 2AB-derivatives of di- and oligo-saccharides were analyzed by anion-exchange HPLC on a PA-G column (YMC Co.)69 (link),70 (link). Unsaturated disaccharides in the digests were identified by comparison with the elution positions of authentic 2AB-labeled disaccharide standards and newly prepared 3-O-sulfate-containing oligosaccharides.
Ultrasonic homogenizer
Manufactured by TAITEC
Sourced in Japan
The Ultrasonic homogenizer is a laboratory instrument designed to homogenize, disperse, and emulsify samples through the application of high-frequency sound waves. It generates ultrasonic vibrations that create cavitation, effectively breaking down particles and mixing the sample components.
Automatically generated - may contain errors
Lab products found in correlation
Biorobot ez1 system, by Qiagen (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Heparinase 2, by R&D Systems (1 mentions)
Pa g column, by YMC (1 mentions)
Amicon ultra 4, by Merck Group (1 mentions)
Sandwich elisa, by Fujifilm (1 mentions)
Flexcontrol 3.0 software package, by Bruker (1 mentions)
Flexanalysis 3, by Bruker (1 mentions)
Ultraflex 2 tof tof mass spectrometer, by Bruker (1 mentions)
Tris 2 carboxyethyl phosphine, by Merck Group (1 mentions)
Iodoacetamide, by Merck Group (1 mentions)
10 protocols using ultrasonic homogenizer
1
Quantifying Heparan Sulfate Disaccharides
2
Modulation of MyD88 Signaling by S1PC
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J774 cells were lysed with deionized water containing protease and phosphatase inhibitor and sonicated using ultrasonic homogenizer (TAITEC, Saitama, Japan). The lysates were centrifuged at 10,000 g for 10 min at 4 °C. The supernatants were collected. Cell lysates were treated with S1PC (0.3, 1 and 3 mM) or SAC (3 mM) for 10-60 min at 37 °C. 293FT cells were transfected with pcDNA3.1+/c-(k) dyk containing MyD88 (Genscript, NJ, USA). After 24 h, cells were lysed with deionized water containing protease and phosphatase inhibitor and sonicated using ultrasonic homogenizer. Recombinant MyD88-DYK was purified using anti-DYKDDDDK tag magnetic beads. Recombinant MyD88-DYK was treated with S1PC (1 and 3 mM) for 60 min at 37 °C.
3
Ceramide Distribution in Mouse Brain
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A 100 μL volume of 0.1 mM or 1 mM each ceramide (or vehicle only for controls) and 2 mM FITC-dextran dissolved in 1% BSA/phosphate-buffered saline (PBS) was injected into BALB/cCrSlc mice via the tail vein. Before injection, we mixed sample solutions to disperse by vortex and sonication, and incubated at 37°C for 10 min. To measure the concentration of ceramides in brain tissues, mice were perfused with PBS containing 5 U/mL heparin at 30 min after injection, and brain tissues were collected.
Isolated brains were suspended in PBS and sonicated with an ultrasonic homogenizer (TAITEC, Saitama, Japan). The concentration of ceramides in brains was measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, and that of FITC-dextran was measured with a Appliskan fluorescence plate reader (Thermo Scientific, Waltham, MA). To analyze the blood profile of the injected ceramides and metabolites, we intravenously injected a 100 μL volume of 1 mM each ceramide under the same conditions as above, then dissected and collected blood from the mouse heart at 30 min after the injection. We obtained serum from the collected blood, and measured the ceramide concentration by LC-MS/MS.
Isolated brains were suspended in PBS and sonicated with an ultrasonic homogenizer (TAITEC, Saitama, Japan). The concentration of ceramides in brains was measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, and that of FITC-dextran was measured with a Appliskan fluorescence plate reader (Thermo Scientific, Waltham, MA). To analyze the blood profile of the injected ceramides and metabolites, we intravenously injected a 100 μL volume of 1 mM each ceramide under the same conditions as above, then dissected and collected blood from the mouse heart at 30 min after the injection. We obtained serum from the collected blood, and measured the ceramide concentration by LC-MS/MS.
4
Glycoprotein and FNG Extraction
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For the N-glycan and FNG analyses, glycoproteins and FNGs were extracted as previously described [17 (link)]. Approximately 1 × 106 cells were suspended in Tris-acetate buffer containing 2% sodium dodecyl sulfate and homogenized using an Ultrasonic Homogenizer (Taitec, Saitama, Japan). Reductive alkylation of the cellular proteins was performed, followed by the precipitation of proteins in the presence of a four-fold volume of ice-cold ethanol. The precipitates including proteins were dried, dissolved in ammonium bicarbonate, and digested with trypsin. Finally, N-glycans were prepared by PNGase F difestion. The supernatants containing FNG were completely dried and dissolved in deionized water. Then, the samples were directly subjected to the glycoblotting procedure. Detailed procedures and are provided elsewhere [26 (link)].
5
Quantification of Amyloid-Beta Peptides
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Aß1-40 and Aß1-42 levels were determined with a sandwich ELISA (FUJIFILM Wako). Mouse brains were homogenized in 4 M guanidine-HCl buffer (pH 8.0) with an ultrasonic homogenizer (TAITEC, Saitama, Japan). The homogenates were incubated at room temperature for 3 h, diluted in 0.1% BSA in PBS, and centrifuged at 16,000 × g for 20 min. The supernatants were collected and used for the ELISA. Mouse sera were used directly in the ELISA, and all samples used were measured in duplicate.
6
Glycosphingolipid Glycan Analysis
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The harvested culture cells and the EVs were suspended in acetate buffer (50 mM, pH 5.5) containing 0.2% Triton X-100 and sonicated with an Ultrasonic Homogenizer (TAITEC, Saitama, Japan). To release GSL-glycans, 5 μL of endoglycoceramidase I (EGCase I) from Rhodococcus equi was added to their lysates of cultured cells and their EVs corresponding to 40 μg protein47 (link). EGCase digestion was performed at 37 °C for 16 h. After deglycosylation, ethanol was added to the reaction mixture and incubated at −30 °C for 4 h. For recovery of GSL-glycans, the supernatant fractions were separated by centrifugation and dried with a centrifugal evaporator. The concentrated supernatant containing GSL-glycans were resuspended in 50 μL of H2O and subjected to a glycoblotting procedure as previously described50 (link). The analysis of GSL-glycans was performed by MALDI-TOF MS using an Ultraflex II TOF/TOF mass spectrometer, which was controlled by the FlexControl 3.0 software package (Bruker Daltonics, Bremen, Germany). All spectra were obtained as positive ions and masses were annotated using the FlexAnalysis 3.0 software package (Bruker Daltonics). The SphinGOMAP (http://www.sphingomap.org/ ) online databases were used for structural identification of GSL glycans51 (link).
7
N-Glycan Analysis of Cellular Proteins
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For N-glycan analysis, (glyco)proteins were extracted as described [21 (link)]. Briefly, cell pellets consisting of 1 × 106 cells were homogenized using an Ultrasonic Homogenizer (Taitec Corp., Saitama, Japan) in 100 mM Tris-acetate buffer (100 μL, pH 7.4) supplemented with 2% sodium dodecyl sulfate as a surfactant for the complete dissolution of cell pellets. Reductive alkylation of the cellular proteins was performed by the addition of 500 mM Tris(2-carboxyethyl)phosphine (Sigma-Aldrich) at room temperature for 60 min, followed by the addition of 200 mM iodoacetamide (Sigma-Aldrich) at room temperature for 30 min. After reductive alkylation, ethanol precipitation was carried out adding a 4-fold volume of cold ethanol and incubation for 3 h at −30°C. Supernatants and precipitated proteins were separated by centrifugation at 20,000 × g for 10 min at 4°C, and precipitates were again washed with cold ethanol. Collected precipitates containing glycoproteins/N-glycans were dried at 37°C for 10 min.
8
RNA Extraction from Minced Tissue
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The collected tissues were minced with surgical scissors, soaked in 1 mL TRIzol reagent (Invitrogen), and sonicated by using an ultrasonic homogenizer (Taitec, Saitama, Japan). Two hundred microliters of chloroform was added, and after thorough shaking, the mixture was centrifuged at 22,000 g for 15 min at 4°C. The aqueous layer was transferred to another tube, and total RNA was extracted by the acid guanidiniumthiocyanate-phenol-chloroform method and cleaned up with a BioRobot EZ1 system (QIAGEN, Hilden, Germany), which enables fully automated extraction and purification of nucleic acids by magnetic bead technology. The purity of RNA was assessed by determining the ratio of light absorption at 260 nm (A260) to that at 280 nm (A280). An A260/A280 ratio in the 1.9–2.1 range was considered acceptable. The RNA concentration was determined from A260.
9
Comparative Glycomic Profiling of Pluripotent Cells
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Cultured ESCs and EpiLCs (> 1 × 106 cells) were washed 5 times with PBS and collected using a scraper. Collected cells were resuspended in 100 mL of PBS and homogenized using an Ultrasonic Homogenizer (TAITEC, Saitama, Japan). Cell lysates were precipitated with EtOH and subsequently the proteinous pellet and supernatant fractions were separated by centrifugation22 (link),74 (link),75 (link). The resulting pellet was dissolved in H2O and cellular protein concentration was measured using a BCA protein assay kit (Thermo Fisher Scientific). The pellet fractions corresponding to 25 μg, 50 μg, and 100 μg of proteins were used for N-glycans, O-glycans, and GAG analyses, respectively. The supernatant fraction corresponding to 100 μg of proteins was concentrated for GSL and FOS analyses. Glycomic analyses of N-glycans, GSL, and FOS were performed by glycoblotting methods combined with the SALSA procedure76 (link),77 (link). O-glycome analysis was performed by β-elimination in the presence of pyrazolone analogues (BEP) method, and GAG were measured by HPLC as previously described21 (link). This methodology allows a comparative analysis of glycomes. The deduced composition and absolute amount of detected structures is listed in Supplementary Table S4 .
10
Total RNA Extraction Protocol
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The collected tissues were minced with surgical scissors, soaked in 1 ml TRIzol Reagent (Invitrogen), and sonicated by an ultrasonic homogenizer (Taitec, Saitama, Japan). Two hundred μl chloroform was added, and after thorough shaking, the mixture was centrifuged at 22,000 xg for 15 min at 4 °C. The aqueous layer was transferred to another tube, and total RNA was extracted by the acid guanidiniumthiocyanate-phenol–chloroform method and cleaned up with a BioRobot EZ1 system (QIAGEN, Hilden, Germany), which enables fully automated extraction and purification of nucleic acids by magnetic bead technology. The purity of RNA was assessed by determining the ratio of light absorption at 260 nm (A260) to that at 280 nm (A280). An A260/A280 ratio in the 1.9–2.1 range was considered acceptable. The RNA concentration was determined from A260.
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