Vnano, L-Vnano, Enano, and L-Enano were synthesized, and the formulation screening was conducted to optimize L-Enano components as we previously reported [25 (link)]. The optimal formulation by the weight ratio of PC/Kolliphor® HS15/(+)-α-tocopherol acetate was 1:1.25:0.25. Enano was synthesized using 73 mg lipid mixture containing 10% EGCG, 36.2% soy PC, 45% surfactant Kolliphor® HS15, and 8.8% (+)-α-tocopherol acetate (all in weight %). The lipid mixture in ethanol was dried under nitrogen to form a lipid film. Enano was produced by reconstituting the dried lipid film in 10 mL deionized water followed by homogenization (PowerGen 125, Fisher Scientific, Pittsburgh, PA) for 2 minutes and sonication for additional 2 minutes using a Branson Sonifier S-450. L-Enano was made by replacing 30 mol% soy PC with KOdiA-PC. Vnano and L-Vnano were synthesized by the same procedures and components without adding EGCG. We measured particle size and polydispersity index of nanoparticles using a BI-MAS particle size analyzer, and their zeta potential using a ZetaPALS analyzer (Brookhaven Corporation, Holtsville, NY, USA). For primary mouse peritoneal macrophage binding and uptake experiments, fluorescence NBD- PC was incorporated into nanoparticles by replacing 2 mol% total PC to make NBD-labeled nanoparticles.
Sonifier s 450
Manufactured by Emerson
The Sonifier S-450 is a lab equipment product designed for ultrasonic cell disruption and homogenization. It generates high-frequency sound waves to break down samples, such as cells or tissues, for further analysis or processing.
Automatically generated - may contain errors
Lab products found in correlation
Nextseq platform, by Illumina (3 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (3 mentions)
Sureprep soil dna isolation kit, by Thermo Fisher Scientific (3 mentions)
Powergen 125, by Thermo Fisher Scientific (1 mentions)
Cup horn, by Emerson (1 mentions)
Coomassie staining, by Merck Group (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
7 protocols using sonifier s 450
1
Characterization and Optimization of Nano-Enabled Drug Delivery Systems
2
Extraction and Purification of Gangliosides
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The total gangliosides were extracted from transfected CHO cells by mixing 107 cells with 3 mL chloroform/methanol (1:2, v/v) and sonic dispersion. After twenty pulses being given by a Sonifier S-450 equipped with a cup horn (Branson), the samples were incubated for 15 min. in a bath sonicator. Debris was removed by centrifugation (1600× g for 10 min.) and the supernatant was transferred into a new tube. After adjusting a final ratio of chloroform/methanol/water of 4:8:5 (v/v/v), the samples were centrifuged (1600× g for 10 min.) and the upper phase containing the ganglioside fraction was desalted on a Chromabond C18 column (Macherey-Nagel, Düren, Germany). The gangliosides were dried under a nitrogen stream, dissolved in 20 µL of chloroform/methanol (1:2, v/v) and stored at −20 °C.
3
Nanoparticle-Protein Complex Isolation
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Samples were centrifuged 1 h at 14 100g (Mini-centrifuge MiniSpin Plus, VWR International, Fontenay-sous-Bois, France) at room temperature to separate the nanoparticle–protein complexes from the cell culture medium. The supernatant was discarded and the pellets were washed with 1 mL PBS to remove the proteins that did not bound tightly to the nanoparticles, the tubes were sonicated (Branson Sonifier S-450, amplitude 89%, pulse 2 s and intervals 2 s). Two additional washes consisting of a 20 min centrifugation at 14 100g, room temperature were carried out. The supernatant was finally discarded and the pellet containing the nanoparticle–protein complexes was used for the next step.
4
Membrane Fractionation of Coxiella and E. coli
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For Coxiella membrane fractionation, 100 ml of wt Coxiella or Tn208 mutant grown in ACCM-2 for 7 days were pelleted and resuspended in 200 µl 20 mM Tris pH 8 containing 1× Complete protease inhibitor (Roche). For E. coli membrane fractionation, 30 ml of IPTG-induced or non-induced E. coli BL21-DE3 star pLysS pET27b-OmpA, pET27b-OmpAΔL1, pET27b-OmpAΔL2, pET27b-OmpAΔL3 or pET27b-OmpAΔL4 were pelleted and resuspended in 5 ml 20 mM Tris pH 8 containing 1× Complete protease inhibitor (Roche). Bacteria were sonicated using a Branson Sonifier S-450 (6 pulses of 20 s at 40% intensity) and cleared by centrifugation at 10000 g for 5 min at 4°C. Inner membrane proteins were extracted by incubation with sarkosyl (0.5% final concentration) at RT for 15 min. Outer membrane proteins were pelleted by ultracentrifugation (TLA-100, 32000 r.p.m., 30 min, 4°C) and resuspended in 2× Laemmli sample buffer. Insoluble, soluble/sarkosyl-solubilized and outer membrane fractions were resolved by SDS-PAGE and analyzed by Coomassie staining (Sigma) or immunoblotting with anti-OmpA and anti NMII antibodies.
5
Genomic DNA extraction and sequencing of Frankia
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Frankia strains AgB32 and AgKG'84/4 were grown from stocks preserved in 20% vol/vol glycerol at -80 °C since 2003 in Defined Propionate Medium (DPM) containing propionate and NH4Cl as C and N source, respectively (19), at 30 °C for two weeks. Cells were harvested by centrifugation (15,000 × g, 5 min) and homogenized through sonication (10 s at 20% output in a S-450 sonifier, Branson Ultrasonics, Danbury, CT) 20 (link). DNA was extracted from cell pellets after an additional centrifugation step using the SurePrepTM Soil DNA Isolation Kit (Fisher Scientific, Houston, TX) 21 (link), and concentrations measured with a Qubit® 2.0 Fluorometer (Life Technologies, Carlsbad, USA). Library preparation and sequencing using the Illumina tagmentation protocol and the NextSeq Illumina platform (2 × 150 bp) using standard protocols were done at the Microbial Genomics Sequencing Center, Pittsburgh, PA, USA.
6
Sequencing Genomic DNA from Frankia Strains
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Frankia strains Ag45/Mut15 and AgPM24 that were previously identified as members of cluster 1, representing a subcluster designated as subgroup II (14) or cluster 1b (12) were from a stock frozen at -20 °C in Defined Propionate Medium (DPM) containing propionate and NH4Cl as C and N source, respectively (15), at 30 °C for two weeks. Cells were harvested by centrifugation (15,000 × g, 5 min) and aggregates homogenized by brief sonication (10 s at 20% output in a S-450 sonifier, Branson Ultrasonics, Danbury, CT) (16). After an additional centrifugation, DNA was extracted from cell pellets using the SurePrepTM Soil DNA Isolation Kit (Fisher Scientific, Houston, TX) (17). DNA concentrations were measured with a Qubit® 2.0 Fluorometer (Life Technologies, Carlsbad, USA), and DNA sent to the Microbial Genomics Sequencing Center, Pittsburgh, PA, USA for library preparation and sequencing using the Illumina tagmentation protocol and the NextSeq Illumina platform (2 × 150 bp) using standard protocols.
7
Genome Sequencing of Frankia Strains
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Seven Frankia strains previously identified to represent members of cluster 4 (AgW1.1, AgB1.9, AgB1.8, CN4, CN6, CN7, CNM7) were grown in Defined Propionate Medium (DPM) containing propionate and NH4Cl as C and N source, respectively 29 , at 30°C for two weeks. Cells were harvested by centrifugation (15,000 x g, 5 min), and cell aggregates homogenized by brief sonication (10s at 20% output in a S-450 sonifier, Branson Ultrasonics, Danbury, CT) 30 (link). After centrifugation, cell pellets were used for DNA extraction using the SurePrepTM Soil DNA Isolation Kit (Fisher Scientific, Houston, TX) with small modifications as described before 31 (link). Extractions of all samples were done in triplicate, and DNA concentrations measured with a Qubit® 2.0 Fluorometer (Life Technologies, Carlsbad, USA). Library preparation and sequencing was performed at the Microbial Genomics Sequencing Center, Pittsburgh, PA, USA using the Illumina tagmentation protocol and the NextSeq Illumina platform (2 x 150 bp).
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