The in vitro release profile of CUR-NP formulation was estimated using two different dissolution media. The first one corresponded to water (M-1) and the second was phosphate buffered saline of pH 7.4 containing MeOH 20% and 2.5% of Tween 80 (M-2). 1 mL of each formulation was placed in 80 mL of the respective medium maintained at 37 ± 0.5 °C and 150 rpm constant agitation in a Labnet 211 DS shaking incubator. A volume of 4 mL of each solution were withdrawn at specific time intervals without replacing the volume. The aliquots were centrifuged at 6000 rpm for 10 min in a Thermo Scientific Sorvall ST 16R centrifuge maintaining at 37 °C. The concentration of CUR was determined using a spectrophotometer Shimadzu 1800 double beam UV-Vis at a wavelength of 420 nm. Dissolution profiles of free CUR was evaluated in both dissolution media for comparison with CUR release from the developed NP. The sampling was done in triplicate.
Sorvall st 16r centrifuge
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The Sorvall ST 16R Centrifuge is a compact, refrigerated benchtop centrifuge designed for a wide range of applications in the laboratory. It features a maximum speed of 16,000 rpm and a maximum RCF of 25,062 x g. The centrifuge is equipped with a brushless, maintenance-free motor and has a user-friendly control panel with a digital display for quick settings and monitoring of the run.
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Lab products found in correlation
Multiskan go microplate spectrophotometer, by Thermo Fisher Scientific (7 mentions)
Sonifer 450, by Emerson (2 mentions)
Fetal bovine serum (fbs), by System Biosciences (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Hplc grade water, by Thermo Fisher Scientific (1 mentions)
Ultimate 3000 autosampler, by Thermo Fisher Scientific (1 mentions)
Picofrit emitter, by New Objective (1 mentions)
Formamide, by Promega (1 mentions)
Ultrasonic bath 2800, by Emerson (1 mentions)
Normal human serum, by Merck Group (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
Phytohemagglutinin (pha), by Merck Group (1 mentions)
Pettrace 16.5 mev cyclotron, by GE Healthcare (1 mentions)
Tracerlab fx c pro module, by GE Healthcare (1 mentions)
Crc 25r, by Mirion Technologies (1 mentions)
Gc 2010 plus, by Shimadzu (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Penicillin streptomycin, by Gemini Bio (1 mentions)
30 protocols using sorvall st 16r centrifuge
1
In Vitro Release Kinetics of Curcumin Nanoparticles
2
Preparation of SARM1 SAM-TIR Protein from Transfected HEK293T Cells
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NRK1-HEK293T cells (Essuman et al., 2017 (link)) were seeded onto 150 cm2 flasks at 10 × 106 cells per plate. The next day, the cells were transfected with 15 μg of human SAM-TIR expression plasmid using X-TremeGENE 9 DNA Transfection Reagent. Human SAM-TIR expression plasmid consisted of a Strep-TEV-human SARM1, aminoacids 408–700, cloned in pSF-CMV-Amp using NcoI and XbaI sites. The cultures were supplemented with 1 mM nicotinamide riboside (NR) at time of transfection to minimize toxicity from SAM-TIR overexpression (Essuman et al., 2017 (link)). Forty-eight hours after transfection, cells were harvested, pelleted by centrifugation at 1,000 rpm (Sorvall ST 16R centrifuge, ThermoFisher), and washed once with cold PBS (0.01 M phosphate buffered saline NaCl 0.138 M; KCl 0.0027 M; pH 7.4). The cells were resuspended in PBS with protease inhibitors (cOmpleteTM protease inhibitor cocktail) and cell lysates were prepared by sonication (Branson Sonifer 450, output = 3, 20 episodes of stroke). The lysates were centrifuged (12,000 × g for 10 min at 4°C) to remove cell debris and the supernatants (containing SARM1 SAM-TIR protein) were stored at −80°C for later use in the in vitro SARM1 SAM-TIR NADase assay (see below). Protein concentration was determined by the Bicinchoninic (BCA) method and used to normalize lysate concentrations.
3
Erythrocyte Hemolysis Assay Protocol
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The assay was conducted according to the procedure reported by Avrahami and Shai [38 (link)]. Briefly, the fresh hRBCs with EDTA as anticoagulant were rinsed three times with PBS through centrifugation at 800× g for 10 min and resuspended in PBS. The serial dilution of peptides (1–512 µg/mL) was prepared in PBS on 96-well plates. Then, the stock RBCs solution was added to the plates to reach a final volume of 100 µL with a 4% concentration of erythrocytes (v/v). The control wells for 0 (RBCs in PBS) and 100% (RBCs in 1% Triton-X 100) hemolysis were also prepared. Then, the plates were incubated for 1 h at 37 °C and centrifuged at 800× g for 10 min at 4 °C (Sorvall ST 16R Centrifuge, Thermo Scientific, Waltham, MA, USA). After centrifugation, the supernatant was carefully transferred to new microtiter plates and the release of hemoglobin was measured at 540 nm (Multiskan™ GO Microplate Spectrophotometer, Thermo Scientific, Waltham, MA, USA). The percentage of hemolysis was calculated based on wells with 100% hemolysis.
4
Extracellular Vesicle Purification and Cryopreservation
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The conditioned cell culture media (typically 800 mL) were centrifuged at 800× g for 30 min (Sorvall ST 16R centrifuge, Thermo Scientific, Grand Island, NY, USA) to remove cellular debris. A KrosFlo Research 2i Tangential Flow Filtration System (Spectrum Labs, Los Angeles, CA, USA) was used to concentrate and purify EVs, as previously described [55 (link),56 (link)]. Briefly, supernatants from cell culture media were filtered through sterile and rehydrated hollow fiber polyethersulfone membranes (0.65 μm pores), and the permeate was further filtered through sterile and rehydrated hollow fiber polysulfone membranes (500 kDa molecular weight cutoff). The final retentate was diafiltrated six times with a clinical-grade cryoprotective buffer (5% sucrose, 50 mM Tris, and 2 mM MgCl—Lonza, #08-735B, Bend, OR, USA) [24 (link)] and concentrated to a final volume of 6–9 mL for functional studies. For GNA, diafiltration was performed six times in high performance liquid chromatography (HPLC)-grade water (Thermo Fisher Scientific, Waltham, MA, USA). Aliquots of 500 μL were prepared in low protein binding microtubes and stored at −80 °C until further analysis.
5
Stability and Bioactivity of Optimized Emulsion
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Stability profiles of the obtained emulsion containing the optimized extract combination and the emulsion base were evaluated in terms of physical stability and biological properties after storing under various conditions including room temperature with light (RTL) and without light (RTD), 4 °C, 45 °C for three months, and accelerated condition as 6 cycles of heating–cooling (HC). After storing, viscosity and pH value of each formulation were measured and compared with those initially. In addition, DPPH scavenging effect of the extract-loaded formulation was determined and compared with those initially. To evaluate the biological properties, 10% Tween 20 served as an extracting solvent for the extract-loaded formulation in a ratio of 1:1. The mixture was sonicated for 30 min and centrifuged at 10,000 rpm for 30 min (Model: Sorvall ST16R centrifuge, Thermo Fisher Scientific, Osterode am Harz, Germany). Supernatant was then collected and used for the evaluation.
6
DEHP Analysis by ESI-MS/MS
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DEHP was obtained from Sigma-Aldrich (St. Louis, MO, USA). Formamide was purchased by Promega (Madison, WI, USA). ReproSil-Pur C4 size 5 μm stationary phase was supplied by Dr. Maisch GmbH (Ammerbuch-Entringen, Germany). Chromatography columns were obtained from Polymicro Technologies (Phoenix, AZ, USA) and PicoFrit Emitter were obtained from New Objective (Woburn, MA, USA). The Sorvall ST 16R Centrifuge was supplied by Thermo Scientific (Waltham, MA, USA). The UltiMate 3000 autosampler was purchased from Dionex (Ottawa, ON, Canada) and the AB Sciex QTRAP 4000 ESI-MS/MS Hybrid Triple Quadrupole/Linear Ion Trap was purchased from AB Sciex (Framingham, MA, USA).
7
Low CRP Atherothrombosis Risk Protocol
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We enrolled patients with low CRP concentration with atherothrombosis risk treated in the Cardiology Department of Beijing Tsinghua Changgung Hospital from December 2018 to March 2019 in which either wr‐CRP or hs‐CRP was assayed, and excluded those with CRP ≥ 20 mg/L. Each participant was enrolled once and 200 cases met our criteria. Blood samples were collected in heparin‐lithium anticoagulated tubes (Vacuette Greiner, ref#474084) and analyzed within 4 hours after blood is withdrawn. All tubes were centrifuged on Sorvall ST 16R centrifuge (Thermo Scientific) for 10 min at 2000 g (temperature 19.0 ± 0.4°C).
8
Preparation of Polyelectrolyte Complex Coacervates
9
Preparation of SARM1 SAM-TIR Protein from Transfected HEK293T Cells
10
Serum Stability of Peptides B7 and C7
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Serum stability of peptides B7 and C7 was tested in normal human serum (Sigma-Aldrich, Steinheim, Germany) according to the modified Jenssen and Aspmo protocol [145 (link)]. Briefly, compounds were incubated in 25% (v/v) human serum solution in RPMI-1640 (Sigma-Aldrich, Steinheim, Germany) at 37 ± 0.1 °C. The final compound concentration was 50 µg/mL. Time intervals were 0, 1, 2, 3, 4, and 5 h. To precipitate serum proteins, 100 µL of solution was added to 200 µL of 96% ethanol (Pure P.A., POCH, Avantor Performance Materials S.A, Gliwice, Poland) and incubated at 4 °C before centrifugation (4 °C, 18,000× g, 2 min; Sorvall ST 16R Centrifuge, Thermo Scientific, Osterode am Harz, Germany). Subsequently, 200 µL of supernatant was transferred to glass vials (1.5 mL, Anchem, Poland), and the liquid was evaporated under nitrogen at 37 °C (approximately 30 min). The vials were lyophilized for 24 h to remove traces of solvents.
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