Comminuted samples of apple, kiwi, carrot, kale, orange, black olive, pork loin, salmon, and avocado (15 g) were individually weighed in 50 mL polypropylene tubes along with 7.5 g HCO2NH4, which was extracted for 10 min with 15 mL MeCN using a Glas-Col (Terre Haute, IN; USA) platform pulse mixer at 80 % setting with maximum pulsation. For wheat grain and dried basil, 5 g sample + 15 mL water + 7.5 g HCO2NH4 was added to the tubes with 15 mL MeCN and extraction time was 60 min using the platform shaker (capacity of 50 tubes at a time). For reagent blanks, 15 mL water represented the sample. Then, centrifugation at 4150 rpm (3711 rcf) at room temperature for 3 min was conducted using a Thermo Fisher (Waltham, MA; USA) Sorvall Legend RT centrifuge (capacity of twenty 50 mL tubes at a time). Extracts of individual matrices were combined and spiked (or not) with the analytes and IS to evaluate the automated mini-SPE cleanup step. The initial extracts (spiked or not) were transferred to 1.8 mL standard ambler glass autosampler (AS) vials, which were closed with split septa caps.
Sorvall legend rt centrifuge
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Sorvall Legend RT centrifuge is a benchtop refrigerated centrifuge designed for high-performance sample processing in the laboratory. It features a robust and durable construction, with a maximum speed of 17,500 rpm and a maximum RCF of 30,130 x g. The centrifuge is equipped with temperature control capabilities, allowing users to maintain consistent sample conditions during the centrifugation process.
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Lab products found in correlation
Centrifuge 5810r, by Eppendorf (2 mentions)
Amicon ultra centrifugal filter, by Merck Group (2 mentions)
Taqman gene expression master mix, by Thermo Fisher Scientific (1 mentions)
Custom taqman gene expression array card, by Thermo Fisher Scientific (1 mentions)
Viia 7 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Dulbecco phosphate buffered saline (dpbs), by Cytiva (1 mentions)
Optiprep, by Merck Group (1 mentions)
Propidium iodide, by Promega (1 mentions)
Modfit lt software, by Verity Software House (1 mentions)
Facscan flow cytometer, by BD (1 mentions)
10 protocols using sorvall legend rt centrifuge
1
Automated Extraction and Cleanup for Food Matrices
2
Platelet-rich Plasma Preparation
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WB bags were allowed to stand at 22 °C for one hour after collection. The PRP method was applied to obtain the PC as follows: each WB bag was subjected to light centrifugation (1600 X g) for 6 min at 22 °C (Sorvall Legend RT + Centrifuge, Thermo Scientific, Waltham, USA). PRP was removed using a manual plasma extractor (ACS201, Terumo Medical of Brazil, São Paulo, BRA) and then subjected to a second centrifugation (3300 X g) for 8 min at 22 °C. Fluid weight and density (1.026 g/mL) were used to calculate the final volume of the bag [26 (link)]. Excessive plasma was removed with the aid of a plasma extractor until 50–70 mL of residual plasma remained at the bottom of the bag with the sedimented platelets [25 ]. PC were stored in polyvinyl chloride (PVC) bags plasticized with tri-2-ethyl-trimellitate (TOTM, JP Indústria Farmacêutica), which are specific for platelet storage and do not have anticoagulants nor additive solutions.
3
Custom TaqMan Gene Expression Analysis
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Customised TaqMan low-density array (TLDA) 384-well microfluidic cards with inventoried or made-to-order predesigned assays were designed for our selected 95 genes using the Custom TaqMan Gene Expression Array Card service from Life Technologies, Carlsbad, California, USA (format 96a). For each reservoir, around 700 ng of the purified cDNA was mixed with 100 µL of 2X TaqMan Gene Expression Master Mix (Applied Biosystems, Carlsbad, California, USA) and applied to each fill-port in a 200 µL final volume mix. Using the provided swing-buckets, the TLDA cards were centrifuged in order to fill the wells during two 1 min rounds at 1200 rpm each in a Sorvall Legend RT centrifuge (Thermo Scientific). After cutting off the reservoir flap, the TLDA cards were submitted to perform in the ViiA 7 real-time PCR System (Applied Biosystems). Samples were heated at 50°C for 2 min, at 95°C for 10 min and were then submitted to 50 cycles of 95°C for 15 s and 60°C for 1 min. After the real-time PCR run, the cycle threshold (Ct) of the genes that did not amplify (specified by the ViiA 7 software as ‘undetermined’) was arbitrarily assigned a value of 50 (which refers to the number of PCR cycles).
4
Detergent-free Mitochondria Purification
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Purification of mitochondria without detergent was performed as described previously [87 (link)]. Briefly, 2 × 15 cm fully confluent plates for each stable cell line or the parental cell lines were harvested by trypsinization and washed once with PBS) at 215×g in a swinging bucket table top centrifuge (Sorvall legend RT + centrifuge, ThermoScientific) for 5 min at 4 °C. Cells were gently resuspended in 7.5 ml of mitochondria isolation buffer (MIB; 20 mM Hepes/KOH, pH 7.5, 0.25 M sucrose, 1 mM EDTA, 1 mM DTT, 0.1 mg/ml BSA, 1 x PIM, 1 mM PMSF) and passed once through a 1.5 inch, 25 gauge needle directly into a 15 ml Dounce homogenizer with 0.0010–0.0030 inch clearance. They were homogenized with 25 strokes, diluted with 7.5 ml of MIB and centrifuged at 2000×g, 5 min, 4 °C. The supernatant was saved separately, and the pellet subjected to homogenization once more. The collected supernatants were centrifuged at 10,000×g, 12 min, 4 °C. The pellet from the high-speed spin is the crude mitochondrial fraction. It was resuspended in MIB with 10% DMSO, flash frozen in liquid nitrogen and stored at 80 °C until use.
5
Bifidobacterium infantis Secretory Fractions
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Bifidobacterium longum subsp infantis (B. infantis), obtained from ATCC (Manassas, VA) (ATCC No. 15697), were cultured anaerobically in a media modified from the combination of Mann-Rogosa-Sharpe (MRS) broth (DIFCO; BD Bio- science, Franklin Lakes, NJ) and H4 cell culture media (Supplemental TABLE S1 ) (8 (link),18 (link)). B. infantis conditioned media at the stationary growth phase was prepared by centrifugation of probiotic cultures at 3700 rpm (equal 2936g) (Sorvall legend RT+ centrifuge, ThermoFisher Scientific, MA) for 10 min at 4°C and then by use of 0.22-μm filtration to eliminate residual bacteria. The tested filtrate was used for B. infantis secretory fractions (SFs) separation by high speed centrifugation (3220g, 30 min at 4°C) (Eppendorf centrifuge 5810R, Eppendorf North America, NY) with different sizes of the Amicon Ultra centrifugal filters (MilliporeSigma, MA). The secretory fractions (<3KD, 3–10KD and >10KD) were used to test anti-inflammatory effects in H4 cells before subjecting them to the characteristic identification.
6
Virus-Like Particle Purification
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Five days post-transfection, cell suspensions (viability ≥95%) were centrifuged for 15 min at 3200 rpm on a Sorvall legend RT centrifuge (Thermo Fisher Scientific). Cell supernatants were then treated with 10 U/mL of Denarase (C-LEcta), 5 mM of MgCl2, and incubated for 1 h at 37 °C. For VLP sedimentation, treated supernatants were centrifuged over a 15% Optiprep (w/v) (Sigma-Aldrich)19 (link) cushion (10% of total volume) at 5300 × g for 16 h at 4 °C. After centrifugation, supernatants were discarded and pellets resuspended in DPBS (Cytiva).
7
Bifidobacterium infantis Secretory Fractions
8
Dietary Fat Composition Analysis
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Diets were identical with the exception of the FA composition. All fat blends, except for the addition of fish oil, were prepared in-house. The U.S. fat blend was designed of lard, walnut oil, high-oleic sunflower oil, coconut oil, and palm oil in a ratio of 18.8:3.6:2.8:1.8:1.0 to reflect the FA profile of an average U.S. American diet [39 ] (Table 4 ). Pure butter fat was separated from water through gentle heating of butter into a liquid phase and subsequent centrifugation at 3,434 g at 4°C for 60 min (Sorvall Legend RT Centrifuge, ThermoFisher Scientific, Waltham, MA, USA) to yield a solid supernatant of pure butter fat. Echium oil was procured from Technology Crops International (Winston-Salem, NC, USA). Dairy fat and echium oil replaced 30% (by weight) of U.S. fat blend to produce BO and EO fat blends, respectively. Fat blends were sent to Research Diets, Inc. (Brunswick, NJ, USA) to be incorporated into the diets (pelleted form). In the case of the fat blend supplemented with fish oil, menhaden oil (30% final weight) was blended into the U.S. fat blend on-site at Research Diets, Inc. All experimental diets were isoenergetic and consisted of 17% protein, 43% carbohydrates, and 40% fat (Table 4 ). The complete FA composition of the experimental fat supplements is shown in S6 Table .
9
Extraction and Analysis of TXT-NP
10
Cell-Cycle Analysis of Combination Treatments
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