For Pluronic–clay composites, nanoclay particles were simply added to the Pluronic formulation under magnetic stirring at 4 °C (ice bath) until a homogeneous suspension was obtained. To prepare methylene blue intercalated clays (clay-MB hybrids), a 2 wt.% aqueous clay suspension was first prepared under magnetic stirring in the appropriate solvent for 12 h, followed by 30 min sonication (if required), and another 12 h left alone to swell and expand. Then, a determined pre-dissolved (in the same solvent) amount of MB (depending on the pretended clay to MB ratio) was slowly added to the clay suspension at room temperature, followed by 24 h mixture stirring. Using clays Na116 and C10A, three weight ratios were tested: 20:1, 4:1, and 2:1. Given the results obtained, the remaining clays were mixed with MB using a 2:1 weight ratio. At the end of the process, the clay-MB hybrid’s/MB-loaded clay’s solid phase was separated from the liquid phase by centrifugation using a Heraeus Multifuge X1R Centrifuge from Thermo Fisher for 20 min at 10,000 rpm. The solid pellet phase was washed a couple of times. The centrifugation resultant clay-MB pellet was freeze-dried (VaCo 2, Zirbus Technology) or dried at room temperature [36 ].
Heraeus multifuge x1r centrifuge
Manufactured by Thermo Fisher Scientific
Sourced in Germany, United States
The Heraeus Multifuge X1R centrifuge is a compact, high-performance laboratory centrifuge. It features a versatile rotor design and intuitive touchscreen controls. The centrifuge is capable of separating samples at high speeds to facilitate various research and diagnostic applications.
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Lab products found in correlation
Chemidoc xrs imaging system, by Bio-Rad (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Bicinchoninic acid protein assay kit, by Dingguo (2 mentions)
Dmem f 12 glutamax, by Thermo Fisher Scientific (1 mentions)
Trypan blue dye, by Merck Group (1 mentions)
Hepes, by Merck Group (1 mentions)
Histopaque 1077, by Merck Group (1 mentions)
Fetal calf serum (fcs), by Bio&Sell (1 mentions)
T4174, by Merck Group (1 mentions)
Enhanced chemiluminescence, by Bio-Rad (1 mentions)
Xhf dy, by Ningbo Scientz Biotechnology (1 mentions)
Ep 1 econo pump, by Bio-Rad (1 mentions)
Q125 sonicator, by Qsonica (1 mentions)
Symmetry c18 column, by Waters Corporation (1 mentions)
V 660 spectrophotometer, by Jasco (1 mentions)
11 protocols using heraeus multifuge x1r centrifuge
1
Pluronic-Clay Composites with Methylene Blue
2
Chemical Functionalization of CuO-NPs
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Chemical functionalization of CuO‐NPs was performed by using APTMS.[34] A total of 96 mg of CuO‐NPs were dispersed in 19.2 mL of toluene under ultrasonication (Ultrasonic bath Fisher Scientific model FS20). After 10 min, 240 μL de APTMS and 144 μL Et3N were added dropwise to the mixture under nitrogen atmosphere. The flask was sealed, and it was stirred for 6 h at room temperature. The suspension was centrifuged in a Thermo Scientific Heraeus Multifuge X1R centrifuge for 10 min at 4,500 rpm and the pellet was washed with ethanol three times. The solids obtained were dried at room temperature. The functionalized CuO‐NPs were named as CuO‐NP‐NH2.
3
Quantifying Protein Levels via Western Blotting
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Western blotting analysis was performed to determine protein levels as previously described 17 (link). Briefly, cells were harvested and suspended in lysis buffer containing 50 mM Tris·HCl, pH 7.4, 150 mM NaCl, 0.5% NP-40, 20 mM EDTA, 50 mM NaF, 1 mM Na3VO4, 2 mM PMSF and 1 mM DTT. After 10-minitue incubation on ice, the cell lysates were sonicated briefly and centrifuged at 15,000 ×g using Heraeus Multifuge X1R centrifuge (Thermo Scientific, Osterode, Germany) at 4°C for 20 minutes. The supernatants were collected and their protein concentrations were measured using Bicinchoninic Acid Protein Assay Kit (Dingguo Changsheng Biotechnology, Beijing, China). Cell lysates were then separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA) that were probed using antibodies against different proteins and visualized by using ChemiDoc XRS+ imaging system (Bio-Rad, Hercules, CA, USA) with enhanced chemiluminescence (Dingguo Changsheng Biotechnology).
4
Irinotecan Loading into Liposomal Nanoparticles
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The incorporation of IRN into PM DSPE-PEG was performed as reported by [20 (link)] with some adjustments. Briefly, the PM DSPE-PEG were incubated with an IRN solution for 30 min at 60 °C to allow drug loading at 1 mg/mL concentration. After incubation, the micellar formulations were washed and purified three times using Amicon® 30 kDa devices MWCO (Millipore, Burlington, MA, USA) by centrifugation at 10,000 rpm for 10 min (Heraeus Multifuge X1R-Centrifuge, Thermo Fisher Scientific, Waltham, MA, USA). The washed particles were resuspended in water, obtaining formulations named PM DSPE-PEG IRN.
5
Isolation and Culture of Bone Marrow-Derived Mesenchymal Stem Cells
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MSC were isolated from human BM acetabular reaming of patients undergoing hip arthroplasty surgery after obtaining informed consent of the patient (Ethical approval (187/18)). Briefly, mononuclear cells were collected from BM material by Ficoll (Histopaque®-1077, Sigma-Aldrich, Germany) density gradient centrifugation (150 RCF for 5 min, Heraeus Multifuge X1R Centrifuge, Thermo Fisher Scientific, Germany) and repeatedly washed. To obtain adherent BM-MSC fraction, cells were cultured and further expanded in culture medium (DMEM/F-12 GlutaMAX, 31331-028, Gibco, Germany) supplemented with 10% fetal calf serum (FCS, Bio&Sell, Germany), 1% Pen/Strep (P4333, Gibco), 1% HEPES (H0887, Sigma-Aldrich), and 5 ng/mL fibroblast growth factor (FGF, 100-18C, PeproTech, Germany) at 37°C in a humidified atmosphere and 5% CO2. Culture medium was replaced three times a week. Cells were detached from culture flasks by trypsinization (T4174, Sigma-Aldrich), centrifuged and washed. Cell number and viability were assessed with the trypan-blue dye (93595, Sigma-Aldrich) exclusion test. BM-MSC in passage 4–6 were used for the experiments.
6
Quantitative Protein Expression Analysis
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To determine the protein expression level, we performed Western blotting analysis as previously described (Huang et al., 2014 (link), 2016a (link)). Briefly, cells were harvested, washed and lysed using 50 mM Tris·HCl, pH 7.4, 150 mM NaCl, 20 mM EDTA, 0.5% NP-40, 1 mM Na3VO4, 50 mM NaF, 1 mM DTT and 2 mM PMSF. After 10-min incubation on ice and brief sonication, protein lysates were subjected to centrifugation at 14,000 × g using Heraeus Multifuge X1R centrifuge (Thermo Scientific, Osterode, Germany) at 4°C for 20 min. The concentration of the supernatant was measured by Bicinchoninic Acid Protein Assay Kit (Dingguo Changsheng Biotechnology, Beijing, China). Equal amount of protein lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membrane was then probed with antibodies against different proteins and visualized by using ChemiDoc XRS+ imaging system (Bio-Rad, Hercules, CA, USA) with enhanced chemiluminescence (Dingguo Changsheng Biotechnology). The relative protein levels were determined by the density of Western blot bands as measured by Image J software and normalized against the internal control GAPDH or β-actin.
7
Myofibrillar Protein Extraction from Grass Carp
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MP from Ctenopharyngodon idella was prepared according to the method of Zhang et al. [10 (link)] ith minor modifications. Minced was rinsed and homogenized (XHF-DY, Ningbo Scientz Biotechnology Co, LTD. Ningbo, Xinzhi, China) 5 times with 0.1 mol/L Na3PO4 buffer solution at 3500 r/min followed by centrifugation (Heraeus Multifuge X1R centrifuge, Thermo Fisher Scientific, Osterode, Germany) at 10,000× g at 4 °C for 10 min. The obtained precipitates were homogenized and centrifuged with the same buffer solution twice. After that, the MP pellets were washed twice with five volumes of 0.1 mol/L NaCl using the above-mentioned homogenization and centrifugation conditions. Later on, connective tissues were removed from the MP using 4 layers of cheesecloth. The protein concentration was 75.4 mg/mL determined by the Biuret method using BSA as a standard curve [12 (link)].
8
Postprandial Satiety Hormone Profiling
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A temporary cannula was inserted ~30 min prior to the test meal to allow for the collection of regular blood samples before and after the test meal. Blood samples (~10 mL at each timepoint) were collected at 15 and 5 min before and 15, 30, 45, 60, 120 and 180 min after the consumption of the pasta meal. Blood was collected into vacutainers containing either a clot activator or K2-EDTA anticoagulant and proprietary additives. Serum tubes were left to clot at room temperature for up to 30 min and plasma tubes were stored on ice and centrifuged within 10 min of collection (Heraeus Multifuge X1R centrifuge; Thermo Scientific, Waltham, MA, USA). Plasma was centrifuged at 1900× g for 10 min at 4 °C and serum at 2800× g for 15 min at 4 °C. The resulting serum and plasma were separated into aliquots and stored at −80 °C within 30 min of collection for subsequent analysis of satiety and gut hormones (insulin, ghrelin and glucagon-like peptide-1 (GLP-1)).
9
Synthesis of Calcium Alginate Nanoparticles
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Calcium alginate nanoparticles
were synthesized according to the method described by Daemi and Barikani23 (link) with slight modification. A solution of 0.06%
(w/v) SA was prepared in distilled water under constant agitation
and heated to 60 °C until the polymer was completely dissolved.
Simultaneously, 22 mM CaCl2 solution was prepared by dissolving
in distilled water and titrated into the SA solution at a flow rate
of 0.05 mL/min using a peristaltic pump (EP-1 Econo Pump, Bio-Rad)
under continuous homogenization. The solution was further agitated
for 1 h to achieve the complete formation of the NPs. The homogenized
solution was centrifuged at 15 000g for 10
min (Heraeus Multifuge X1R Centrifuge, Thermo-Fisher Scientific) to
remove impurities. The pellet was reconstituted in 10 mL distilled
water and pulse-sonicated with three cycles of 15 s each at 50% amplitude
(Q125 sonicator, Qsonica) for even dispersion of prepared NPs.
were synthesized according to the method described by Daemi and Barikani23 (link) with slight modification. A solution of 0.06%
(w/v) SA was prepared in distilled water under constant agitation
and heated to 60 °C until the polymer was completely dissolved.
Simultaneously, 22 mM CaCl2 solution was prepared by dissolving
in distilled water and titrated into the SA solution at a flow rate
of 0.05 mL/min using a peristaltic pump (EP-1 Econo Pump, Bio-Rad)
under continuous homogenization. The solution was further agitated
for 1 h to achieve the complete formation of the NPs. The homogenized
solution was centrifuged at 15 000g for 10
min (Heraeus Multifuge X1R Centrifuge, Thermo-Fisher Scientific) to
remove impurities. The pellet was reconstituted in 10 mL distilled
water and pulse-sonicated with three cycles of 15 s each at 50% amplitude
(Q125 sonicator, Qsonica) for even dispersion of prepared NPs.
10
Fatty Acid Analysis by HPLC
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HPLC analysis was performed with a Waters e2695 liquid chromatographic system (Waters, Milford, MA. USA) including a quaternary pump, a 2998 photo-diode array detector (PDA), an automatic sampler and a thermostatic column compartment. A Waters Symmetry C18 column (4.6 mm × 250 mm, particle size 5 μm) was used for the separation of FA extracted in the IL phase. The KQ-500V ultrasonic generator with an electrical power of 500 W was purchased from Kunshan Ultrasonic Instrument Co. Ltd. (Kunshan, China). The centrifugation was carried out on a high-speed freezing Heraeus Multifuge X1R centrifuge from Thermo Scientific (Waltham, MA, USA).
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