In order to evaluate the effect of cinnamon samples on the activity of HepG-2 and BJ-1, cells were seeded in RPMI-1640 medium containing 25 mg/mL of each cinnamon sample, with one 106 cell per flask, and incubated for 48 h at 37 °C under a humidified atmosphere of 5% CO2. After this, the cell medium was converted to serum-free medium (SFM) comprising 10 µL/mL of yogurt extract. Then, trypsin 0.05%/0.53 mM EDTA solutions were used to trypsinize the cell cultures after incubation. After washing in PBS, the cells were centrifuged at 2000 rpm for 5 min at 4 °C and resuspended in 1 mL PBS containing 0.1% Triton X-100. Using a 1.5 mL micro centrifuge tube, cells were sonicated twice for 10 s at 100 Hz (Vibra-cell, Sonics & Material) and centrifuged for 30 min at 4 °C at 14,000 rpm. The cells were sonicated for 2 min at 100 Hz in a 1.5 mL micro centrifuge tube placed on ice, then centrifuged at 14,000 rpm for 30 min at 4 °C after the sonication (Vibra-cell, Sonics & Material). Enzyme activity was tested in the supernatant of the tube. We measured the enzyme activity of SOD, CAT, GSH, and GPx in cell culture according to the manufacturer’s instructions for colorimetric kits ab65354, ab83464, ab142044, and ab102530 Abcam.
Vibra cell
Manufactured by Sonics
Sourced in United States, Switzerland, Germany, United Kingdom
The Vibra Cell is a laboratory instrument designed to disrupt and homogenize samples. It utilizes high-frequency vibrations to efficiently break down and mix materials.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop, by Thermo Fisher Scientific (8 mentions)
Bca kit, by Thermo Fisher Scientific (6 mentions)
Protease inhibitor cocktail, by Merck Group (5 mentions)
Bca assay, by Thermo Fisher Scientific (5 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (5 mentions)
Protease inhibitor, by Merck Group (4 mentions)
Uv vis nir spectrophotometer, by Jasco (4 mentions)
Amicon centrifuge filter, by Merck Group (4 mentions)
Protein coated a g agarose beads, by Santa Cruz Biotechnology (3 mentions)
Zetasizer nano, by Malvern Panalytical (3 mentions)
Dnase, by Thermo Fisher Scientific (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Chromatin immunoprecipitation kit, by Merck Group (2 mentions)
Acetyl histone h3, by Merck Group (2 mentions)
Acetyl histone h4, by Merck Group (2 mentions)
Ab12179, by Abcam (2 mentions)
Ab1012, by Abcam (2 mentions)
Protein a and g sepharose, by Cytiva (2 mentions)
Lysozyme, by Merck Group (2 mentions)
Luminata crescendo, by Merck Group (2 mentions)
300 protocols using vibra cell
1
Cinnamon Antioxidant Activity in HepG2 and BJ-1 Cells
2
Chromatin Immunoprecipitation (ChIP) Protocol
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For ChIP analysis, cells were fixed with 1% formaldehyde for 15 min at room temperature, and then quenched with 2.5 M glycine. Subsequently, the cells were lysed on ice for 10 min in SDS buffer containing protease inhibitors, and then sonicated with Sonics Vibra CellTM (Sonics & Materials Inc., CT, USA; 7 min: 10 s pulse, 10 s interval) on ice. The fragmented chromatin samples were centrifuged at 8000 ×g at 4 °C for 5 min and the supernatant was collected. The samples were pre-cleared, and then incubated overnight at 4 °C with the appropriate antibodies and protein-coated A/G agarose beads (Santa Cruz) with gentle shaking. The immunoprecipitated eluates were reverse cross-linked and recovered through DNA purification for PCR. Anti-H3K4me3 (ab1012; Abcam), anti-H3K9ac (ab12179; Abcam), anti-H3K27me3 (ab6002; Abcam), anti-EZH2 (#5246S; Cell Signaling), anti-SUZ12 (#3737; Cell Signaling), anti-EED (ab96801; Abcam), anti-UTX (ab36938; Abcam), anti-JMJD3 (ab169197; Abcam), and non-immune mouse IgG (sc2025; Santa Cruz) antibodies were used. ChIP-PCR primers are listed in Table 2 .
3
Chromatin Immunoprecipitation with Acetyl-Histone Antibodies
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PFCs were crosslinked with 1% formaldehyde at 37ºC for 10 min, and then glycine was added to a nal concentration of 0.125 M for 5 min to stop the crosslinking. Samples were spun at 800 g at 4ºC for 10 min to pellet tissues. The PFCs were sonicated on wet ice with 300 μl of nuclear lysis buffer using a Sonicator (Sonics Vibra-cell TM , Sonics & Materials Inc., Newtown, Connecticut, USA). The sonication conditions were as follows: almp 70%, pulse 10 sec, pause 59 sec for 9 cycles. The supernatant was collected in accordance with a chromatin immunoprecipitation kit (Millipore, Cat. No. 17408). The lysate was precipitated with antibodies against acetyl-histone H3 (Millipore, Cat. No. 06599) and acetyl-histone H4 (Millipore, Cat. No. 06866). Sequences for ChIP-PCR are as follows: POU3F2: forward primer 5'-CAGTACCGCTGAATAACGC-3' and reverse primer 5'-CTGAGCCTGCTTACGATGA-3'; ANP32A: forward primer 5'-CCATCGGAGCATGTTTCGT-3' and reverse primer 5'-AGGGTGAGATCGCCGTCTT-3'.
4
Chromatin Immunoprecipitation Assay in Prefrontal Cortex
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PFC tissues were crosslinked with 1% formaldehyde at 37°C for 10 min, and then glycine was added to a final concentration of 0.125 M and incubated for 5 min to stop the crosslinking. Samples were centrifuged at 800 g at 4°C for 10 min to pellet tissues. The PFC tissues were sonicated on wet ice in 300 μl of nuclear lysis buffer using a sonicator (Sonics Vibra-cell TM , Sonics & Materials Inc., Newtown, Connecticut, USA). The sonication conditions were used: amplitude 70%, pulse 10 sec, and pause 59 sec for 9 cycles. The supernatant was collected as described in the instructions of a chromatin immunoprecipitation kit (Millipore, Cat. No. 17408). The lysate was precipitated with antibodies against acetyl-histone H3 (Millipore, Cat. No. 06599) and acetyl-histone H4 (Millipore, Cat. No. 06866). The following primer sequences were used for ChIP-PCR: POU3F2: forward primer 5'-CAGTACCGCTGAATAACGC-3' and reverse primer 5'-CTGAGCCTGCTTACGATGA-3'; ANP32A: forward primer 5'-CCATCGGAGCATGTTTCGT-3' and reverse primer 5'-AGGGTGAGATCGCCGTCTT-3'.
The PFC samples used for qPCR and ChIP assay were obtained from the rats in another behavioral experiment, which were different from the samples used in microarray experiments. The samples were processed separately to perform the qPCR and ChIP experiments.
The PFC samples used for qPCR and ChIP assay were obtained from the rats in another behavioral experiment, which were different from the samples used in microarray experiments. The samples were processed separately to perform the qPCR and ChIP experiments.
5
Sonication-Assisted SEA Isolation
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SEA production was achieved according to the method described by Boros and Warren (1970) 26 , modified as follows: the eggs were suspended in ice-cold saline solution and sonicated by using 15 2 min cycles at 30% amplitude (Vibracell, Sonics & Materials, Newtown, CT, USA). Phosphate buffered saline (PBS, 10×, pH 7.4) was added to the material and centrifuged at 30,000 × g for 2h at 4°C. Protein concentration was measured using the Bradford method (Bio-Rad Laboratories, Hercules, CA, USA), and the material was kept at -20°C until use.
6
ATP and ADP Quantification by HPLC
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25 larvae per condition were sonicated (Vibra-cell, Sonics) in ice-cold 0.6 M perchloric acid, and the resulting lysates centrifuged at 3000 × g (10 min, 4 • C). Supernantants were neutralized (pH 7) with KHO, allowed to rest for 30 min on ice, and cleared by centrifugation at 20,000 × g (10 min, 4 • C) to deposit the potassium perchlorate crystals. ATP and ADP were quantified using HPLC with a reversed-phase column [250 × 4.6 mm Luna 5 m C18(2) 100 Å; Phenomenex], a diode-array detector (Agilent 1100 series) and a computer with the Clarity software (DataApex). The elution was performed in isocratic mode, with phosphate buffer (0.06 M K 2 HPO 4 , 0.04M KH 2 PO 4 ; pH 7) at 1.2 mL/min flux. Determinations were performed via 260 nm absorbance, using ATP and ADP standards for calibration curves and specific peak identification.
7
Aqueous Silica Particle Dispersions
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Aqueous dispersions of silica particles were prepared as follows. Particles were dried under vacuum at 170 °C for one hour and were then added to 2.33 mL of water (deionised using a Millipore Milli-Q reagent system; resistivity of at least 18.2 MΩ cm) and dispersed using a pulsed ultrasonic probe (Sonics Vibracell). A typical dispersion protocol involved 1 s of sonication followed by 5 s of cooling, repeated for a total time of 6 min. 0.67 mL of the oil phase, a mixture of dodecane (Sigma-Aldrich, ≥99% - filtered twice through alumina) and isopropyl myristate (Sigma-Aldrich, ≥98% - used as received), was then added prior to the initial emulsification. Interfacial tensions for mixtures of dodecane and isopropyl myristate, and experimentally measured contact angles for this system, are reported in ref. 13 (link). The volume fraction, ΦIM, of isopropyl myristate in the oil phase is used to control the contact angle17 . The particle density was assumed to be 1750 kg m−3, when calculating particle volume fractions, ϕp.
8
Induction of Antifungal Resistance Dynamics
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To determine whether the presence of a sub-inhibitory concentration of antifungal in the medium induces resistance, cultures were seeded over three generations in the presence of antifungals, with a determination of the MIC for each generation. The different strains were cultured in 40 ml of Sabouraud dextrose broth (SBD) medium [21 (link)]. An antifungal (VRC or CIN-102) was added to the culture at a concentration equal to 0, ¼ or ½ of the initial MIC of the strain. After 3 days of incubation at 37 °C with shaking, part of the culture was resuspended in fresh medium containing the same concentration of antifungals and was again incubated at 37 °C for 3 days with shaking, thus forming the second generation. The other part of the culture was homogenized by sonication (Vibra-cell, Sonics and Materials, Inc., Newtown, United States) and then diluted 1 : 10 before MIC determination. These analyses were performed in triplicate and the experiment was performed three times.
To validate the obtained results and to study the induction of resistance over numerous generations, the strains were inoculated on solid SBD medium containing ½ MIC of CIN-102 and were incubated at 37 °C. The strains were sub-cultured onto a new plate every 3 days. After ten generations, the culture was suspended in 0.9 % NaCl, adjusted to 104 cells ml−1, and determination of the MIC was carried out.
To validate the obtained results and to study the induction of resistance over numerous generations, the strains were inoculated on solid SBD medium containing ½ MIC of CIN-102 and were incubated at 37 °C. The strains were sub-cultured onto a new plate every 3 days. After ten generations, the culture was suspended in 0.9 % NaCl, adjusted to 104 cells ml−1, and determination of the MIC was carried out.
9
Purification of Fluorescent Fusion Proteins
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E. coli strain BL21(DE3) was transformed with pET-28a-SH3-I2-GUK::GFP, pET-28a-SH3-I3-GUK::GFP and with pET-28a-CaM::mCherry plasmid DNA. The cells were grown at 37 °C to an optical density of 0.8 at 600 nm and protein expression was induced with 0.5 mM isopropyl-β-d -thiogalactopyranoside ((IPTG) Thermo Fisher Scientific, USA) overnight. The next day, the cells were centrifuged at 4000 x g for 10 min and the pellets were stored at −80 °C. The bacterial cells were lysed by sonication (Vibra cell, Sonics) with Tris buffer (10 mM Tris-HCl [pH 7.4], 150 mM NaCl, imidazole and protease inhibitor (Roche, Switzerland)). As the proteins were predominantly present in the soluble fraction, the sonicated samples were centrifuged at 30,000 x g for 1 h to remove cellular debris. The proteins were purified by Ni2+-nitrilotriacetic acid (NTA)-agarose affinity chromatography. The supernatant was passed through Ni2+-NTA-agarose resin (Qiagen) and the protein-bound beads were washed with 4 column volumes (~25 ml) of wash buffer containing 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 50 mM imidazole. 4 ml elution buffer (10 mM Tris-Cl (pH 7.4), 150 mM NaCl and 500 mM imidazole) was added to elute the target proteins. The eluted proteins were concentrated to 500 μl (Vivaspin-Turbo4, 30 kDa) and was ultracentrifuged at 35,000 x g at 4 °C for 30 min to pellet out the protein aggregates.
10
Immunoprecipitation and Western Blot Analysis
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Co-transfected 293T cells (8 × 105) or virus-infected HF cells were harvested and sonicated in 0.7 ml co-IP buffer [50 mM Tris-Cl (pH 7.4), 50 mM NaF, 5 mM sodium phosphate, 0.1% Triton X-100, containing protease inhibitors (Sigma)] by a microtip probe (Vibra-Cell; Sonics and Materials, Inc., USA) for 10 s (pulse on: l s, pulse off: 3 s). Cell lysates were incubated with appropriate antibodies. After incubation for 16 h at 4°C, 30 μl of a 50% slurry of protein A- and G-Sepharose (Amersham) were added and then the mixture was incubated for 2 h at 4°C to allow adsorption. The mixture was then pelleted and washed 7 times with co-IP buffer. The beads were resuspended and boiled for 5 min in loading buffer. Each sample was analyzed by SDS-PAGE and immunoblotting with appropriate antibodies.
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