In the in vivo experiment, animals were exposed to spherical AgNPs of 20 ± 5 nm in diameter coated with bovine serum albumin (BSA) purchased from PlasmaChem (Berlin, Germany). The preparation of AgNPs was detailed in previous works [14 (link),15 (link)]. To summarize, 2 mg of AgNPs were dispersed in 800 μL of purified distilled water, forming the nanoparticle stock solution. The stock solution was sonicated for 10 min on ice using a probe sonicator (Branson, Danbury, CT, USA) with a total ultrasound energy of 420 J/m. After sonication, 100 μL of 15% BSA and 100 μL of 10× phosphate-buffered saline (PBS) were directly added to the solution. Additionally, the aggregation state of AgNPs was examined alongside the assessment of its zeta potential and hydrodynamic size. The characterization was conducted using scanning electron microscopy (DSM 942, Carl Zeiss, Göttingen, Germany) and transmission electron microscopy (JOEL 1200 EX II, JOEL, Tokyo, Japan). The outcomes of these analyses have previously been published [14 (link)]. The details pertaining to the AgNPs used in the in vivo experiment can be found in Table 1 .
Probe sonicator
Manufactured by Emerson
Sourced in United States
The Probe Sonicator is a laboratory equipment designed for cell disruption, sample preparation, and other applications that require high-intensity ultrasonic energy. It generates high-frequency sound waves to agitate and disrupt samples, effectively dispersing and homogenizing the contents.
Automatically generated - may contain errors
Lab products found in correlation
Kapa hyper prep kit, by Roche (4 mentions)
Ab4729, by Abcam (2 mentions)
Dsm 942, by Zeiss (1 mentions)
Cholesterol quantitation kit, by Merck Group (1 mentions)
Pparγ, by Santa Cruz Biotechnology (1 mentions)
Pierce bicinchoninic acid assay bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
6 well plate, by Sarstedt (1 mentions)
Delsa nano c particle analyzer, by Beckman Coulter (1 mentions)
Ab137676, by Abcam (1 mentions)
Pparγ, by Thermo Fisher Scientific (1 mentions)
Protein a and protein g sepharose, by GE Healthcare (1 mentions)
Diax 900 homogenizer, by Heidolph (1 mentions)
Tween 80, by Merck Group (1 mentions)
Precirol ato 5, by Gattefosse (1 mentions)
Glyceryl monostearate, by Gattefosse (1 mentions)
F3165, by Merck Group (1 mentions)
Complete edta free protease inhibitor cocktail, by Merck Group (1 mentions)
Flag m2, by Merck Group (1 mentions)
Chip seq sample prep kit, by Illumina (1 mentions)
Sephadex g 50 column, by Merck Group (1 mentions)
26 protocols using probe sonicator
1
Characterization and Preparation of BSA-Coated AgNPs for In Vivo Studies
2
Cellular Cholesterol Quantification Assay
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Cellular total cholesterol was measured using cholesterol quantitation kit from Sigma (Cat#MAK043-1KT). In brief, 300k A549, RKO and HFF-1 cells were cultured in 6-well plates and treated with compounds for 20 h at 4 µM. Cholesterol was extracted by adding 200 µL of chloroform:isopropanol:IGEPAL CA-630 (7:11:0.1) and sonicated for 1 min on ice using Branson probe sonicator and 3 s on/off pulses with a 30% amplitude. The samples were centrifuged at 13,000 × g for 10 min to remove the insoluble material. The organic phase was transferred to a new Eppendorf and dried. The lipids were then dissolved in assay buffer. The reaction mix consisting of assay buffer, cholesterol probe, enzyme mix, and cholesterol esterase was added to the samples in 96-well flat-bottom plates. The reactions were incubated for 60 min at 37 °C in the dark and the absorbance was measured at 570 nm.
3
Quantitative Proteomics of Auranofin Effects
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For FITExP experiments, HCT116, A375 and RKO cells were treated with auranofin for 48 h at LC50 concentrations. Methotrexate and paclitaxel were included in these experiments to increase the specificity of detection for differentially expressed proteins. In brief, cells were cultured in 6 well plates at a density of 250 k per well, allowed to detach overnight and treated with the compounds at LC50 concentrations for 48 h. After the treatment period, cells were washed with PBS and lysed with 3% SDC in ambic buffer. Samples were sonicated for 45s, 30% amplitude, 3s on/off cycles using Branson probe sonicator. After total protein quantification using BCA assay, the protein amount in each sample was normalized. DTT was added to a final concentration of 5 mM and samples were incubated for 1 h at room temperature. Subsequently, iodoacetamide was added to a final concentration of 50 mM and samples were incubated in room temperature for 1 h in the dark. The reaction was quenched by adding an additional 10 mM of DTT. After dilution of SDC to 1.5%, digestion was performed at a ratio of 1:100 w/w overnight for Lysyl Endopeptidase. SDC was then diluted to 0.5% and trypsin was added at the same ratio for 6 h. Samples were acidified by TFA, cleaned using SepPak and lyophilized.
4
Chromatin Immunoprecipitation (ChIP) Assay
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ChIP was performed as described previously(Hiraike et al., 2017 (link), 2020 ) with some modifications. Briefly, samples were treated by nuclear extraction buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 0.1% IGEPAL CA-630) for 10 minutes and immediately cross-linked with 1% formaldehyde for 7.5 minutes at room temperature. Cross-linking was quenched using 125 mM glycine for 5 minutes. The chromatin was sheared by a probe sonicator (Branson) and was spun at 15,000 rpm for 5 minutes. Antibodies were added for overnight incubation at 4°C. Mixes of Protein A and Protein G Sepharose (GE) added to samples for 4 hours at 4°C. Subsequent procedures were performed as described previously. The antibodies used were NFI (Santa Cruz Biotechnology, sc-30198) and PPARγ (mix of Santa Cruz Biotechnology, sc-7273, and Perseus Proteomics, A3409A). ChIP-seq libraries were prepared using KAPA hyper prep kit (KAPA Biosystems) according to the manufacturer’s instructions. A list of primers used for ChIP-qPCR analysis is shown in Table S1 D.
5
CNT Dispersion and Sonication Protocol
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Weighing and dispersion were performed as previously described (Arnoldussen et al. 2015 (link)). Briefly, CNTs were weighed and added to dispersion media (DM; (Porter et al. 2008 (link))) before sonication on ice (Branson probe sonicator, 30 % amplitude pulse cycle, 3 × 5 min) and immediate addition to cell culture media.
6
Cell Viability Assay Protocol
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B16–F10 and LLC2 were seeded into 6-well plates (Sarstedt) at a density of 100 000 per well and grown for 24 h. Then cells were treated with IC50 concentration of each drug and corresponding concentration of DMSO (Fig. 1 ). Treatments were conducted in triplicates for each drug and cell lines. After 48 h of treatment, cells were washed two times with PBS, whereupon lysis buffer (1% SDS, 8 M urea, 50 mM Tris pH 8.5) was added on top of the cells. Cells were scraped and collected into tubes and then sonicated using a probe sonicator (Branson) for 45s (3s/3s pulse, 30% amplitude). Protein concentration was measured in each sample using Pierce bicinchoninic acid assay (BCA) protein assay kit (Thermo Fischer Scientific) according to the manufacturer's protocol.
7
Characterization of Nanodiamond Dispersions
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NDs used in this study were sourced from Microdiamant (Switzerland). ND types used were: monocrystalline, synthetic HPHT NDs in 18 nm (0–30 nm, median diameter 18 nm) and 125 nm (0–250 nm, median diameter 125 nm) sizes; monocrystalline, 125 nm NAT NDs (0–250 nm, median diameter 125 nm); and polycrystalline DET ND (cluster size 250–1,000 nm, median 500 nm; individual particle size 4–8 nm). Size specifications were provided by the manufacturer. Air-oxidized NDs were prepared by placing them in a furnace at standard pressure for 1 h at 550 °C after an initial temperature ramp25 . ND samples were mixed with DI water and sonicated with a Branson probe sonicator at 120 W and 50% duty cycle for 40 min to disaggregate ND clusters.
Particle size and zeta potential measurements were performed on ND solutions in a Beckman Coulter Delsa Nano C Particle Analyzer. Particle size measurements confirmed that monocrystalline NDs were well dispersed in water after sonication. Particle sizes of monocrystalline NDs were found to be consistent with manufacturer specifications. DET NDs still displayed some clustering and inconsistent particle size in solution after probe sonication.
Particle size and zeta potential measurements were performed on ND solutions in a Beckman Coulter Delsa Nano C Particle Analyzer. Particle size measurements confirmed that monocrystalline NDs were well dispersed in water after sonication. Particle sizes of monocrystalline NDs were found to be consistent with manufacturer specifications. DET NDs still displayed some clustering and inconsistent particle size in solution after probe sonication.
8
Protein Extraction from Differentiated Cells
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EBs were induced as described above using both iPSCs and ESCs and differentiated for 9 days. iPSCs, hFF, RKO, HT29, and the human neurons differentiated from iPSCs were grown as described in the cell culture section. Then cells were rinsed two time with PBS and lysed in the plate with 1% SDS, 8 M urea, 50 mM Tris buffer pH 8.5. The cells were then sonicated using Branson probe sonicator for 45 s with a 3 s pulse at 30% amplitude. The protein concentration was measured by BCA assay followed by sample preparation for proteomics analysis.
9
Gambogic Acid Loaded Liposomes
10
Chromatin Immunoprecipitation and FAIRE Sequencing
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ChIP was performed as described previously [2 (link)] with some modifications. Briefly, samples were treated by nuclear extraction buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 0.1% IGEPAL CA-630) for 10 minutes and immediately cross-linked with 1% formaldehyde for 7.5 minutes at room temperature. Cross-linking was quenched using 125 mM glycine for 5 minutes. The chromatin was sheared by a probe sonicator (Branson) and was spun at 15,000 rpm for 5 minutes. Antibodies were added for overnight incubation at 4°C. Mixes of Protein A and Protein G Sepharose (GE, for PPARγ andiboty) or Dynabeads Protein A and Protein G (Invitrogen, for FLAG M2, H3K27Ac and KLF5 antibody) were added to samples for 4 hours at 4°C. Subsequent procedures were performed as described previously [2 (link)]. The antibodies used were FLAG M2 (Sigma F3165), PPARγ (mix of Santa Cruz Biotechnology, sc-7273, and Perseus Proteomics, A3409A), H3K27Ac (Abcam ab4729) and KLF5 (Abcam ab137676). FAIRE was performed as described previously [2 (link)]. ChIP-seq as well as FAIRE-seq libraries were prepared using KAPA hyper prep kit (KAPA Biosystems) according to the manufacturer's instructions.
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