Perchloric acid (70%; 25 µl) was added to DNA (50 µl) and incubated in a 95°C water bath for 50 min. KOH (1 mol/l) was used to adjust the pH to between 3 and 5, and the samples were centrifuged at 18,500 × g for 10 min at 4°C, and the supernatant was aliquoted to a clean tube. Methylation levels were determined using an Agilent 1200 High Performance Liquid Chromatography (HPLC) System (Agilent Technologies, Inc., Santa Clara, CA, USA). Separation was achieved with a 150×4.6 mm, 5 µm Agilent Eclipse XDB-C18 column (Agilent Technologies, Inc.) using methanol:water (1:9 v:v) as the mobile phase and a flow rate of 0.3 ml/min. The injection volume was 20 µl, column temperature was 25°C and the detection wavelength was 284 nm. Pure products of 5C and 5mC were used as standards to prepare concentration gradients. External calibration of peak area vs. concentration was used in the determination of global DNA methylation levels: 5mC (%) = [5mC / (5mC + 5C)] × 100%; where 5mC is 5-methylcytosine.
Agilent 1200 high performance liquid chromatography
Manufactured by Agilent Technologies
Sourced in United States
The Agilent 1200 is a high-performance liquid chromatography (HPLC) system. It is designed to separate, identify, and quantify components in a liquid sample. The system includes a solvent delivery module, an autosampler, a column compartment, and a detector. It is capable of performing various HPLC techniques, such as reverse-phase, normal-phase, and ion-exchange chromatography.
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Lab products found in correlation
Agilent eclipse xdb c18 column, by Agilent Technologies (1 mentions)
Autoflex maldi tof mass spectrometer, by Bruker (1 mentions)
Synergy him microplate reader, by Agilent Technologies (1 mentions)
Avance 3 hd 400 nmr spectrometer, by Bruker (1 mentions)
Xevo g2 q tof spectrometer, by Waters Corporation (1 mentions)
Uv 2550 uv vis spectrophotometer, by Shimadzu (1 mentions)
Uv vis detector, by Agilent Technologies (1 mentions)
Wtc 200s5, by Wyatt Technology (1 mentions)
Optilab rex differential refractometer, by Wyatt Technology (1 mentions)
Agilent uv vis diode array detector, by Agilent Technologies (1 mentions)
Kinetex c18 column, by Phenomenex (1 mentions)
6430 series triple quadrupole mass spectrometer, by Agilent Technologies (1 mentions)
Lightcycler 480 2 real time pcr, by Roche (1 mentions)
Elx800 microplate reader, by Agilent Technologies (1 mentions)
7 protocols using agilent 1200 high performance liquid chromatography
1
Quantification of Global DNA Methylation
2
Characterization of Functionalized Magnetic Nanoprobes
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Agilent 1200 high performance liquid chromatography (Agilent Technologies, Inc., Beijing, China), Bruker Avance III 400 HD NMR spectrometer (Bruker, Beijing, China) and Bruker Autoflex MALDI-TOF mass spectrometer (Bruker, Beijing, China) were used to purify and identify the target product, respectively. Bruker TENSOR-27 Infrared Spectroscope (Bruker, Beijing, China), UV-2550 UV–Vis spectrophotometer (Shimadzu Company, Kyoto, Japan) and IONTOF time of flight secondary ion mass spectrometer (ToF-SIMS V) (IONTOF, Munster, Germany) were used to characterize functionalized magnetic nanoprobes. Quantitative mass spectrometry analysis of proteins was performed on a Waters Xevo-G2-Q-TOF spectrometer (Waters, Milford, MA, USA), which was coupled to a Dionex nano-LC3500 System (Thermo Fisher Scientific (China) Co., Ltd., Shanghai, China). SYNERGY HIM Microplate reader (Bio Tek, Winowski, VT, USA) was used to measure the biological activity of baicalin and modified baicalin.
3
HPLC Analysis of Iridoid Glucosides
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Agilent 1200 high performance liquid chromatography (Agilent), photodiode array detector, automatic injector and column temperature box.
The chromatographic conditions based on the method in the literature20 were slightly modified. AE.LICHROM-C18 chromatographic column (250 mm × 4.6 mm, 5 μm, Lanzhou Zhongke Analytical Technology Co., Ltd.). The mobile phase: acetonitrile (A)-0.2% acetic acid (B), gradient elution, 0–8 min, 9%-11% (A); 8–16 min, 11–12% (A); 16–19 min, 12% (A); 19–20 min, 12–9% (A); 20–29 min, 9% (A); the flow rate: 0.8 mL/min. Column temperature was 30 ℃. Detection wavelength: 238 nm. The chromatograph under these conditions is shown in Fig.1 .![]()
The chromatographic conditions based on the method in the literature20 were slightly modified. AE.LICHROM-C18 chromatographic column (250 mm × 4.6 mm, 5 μm, Lanzhou Zhongke Analytical Technology Co., Ltd.). The mobile phase: acetonitrile (A)-0.2% acetic acid (B), gradient elution, 0–8 min, 9%-11% (A); 8–16 min, 11–12% (A); 16–19 min, 12% (A); 19–20 min, 12–9% (A); 20–29 min, 9% (A); the flow rate: 0.8 mL/min. Column temperature was 30 ℃. Detection wavelength: 238 nm. The chromatograph under these conditions is shown in Fig.
HPLC Chromatograms of standards and sample. (
4
Characterization of Macromolecular Samples
5
Phytoextracts Analysis via HPLC-UV
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Qualitative and quantitative analyses of the phytoextracts were carried out using an Agilent 1200 High-Performance Liquid Chromatography coupled with an Agilent UV–Vis diode array detector (Agilent Technologies, Santa Clara, CA, USA). The chromatographic separation was made with a Kinetex C18 column (4.6 × 150 mm2, 5 µm, Phenomenex, Torrance, CA, USA) using several mobile phases and recording UV spectra at different wavelengths [65 (link)], as described in Table 5 .
Each compound was determined comparing the retention times and UV spectra with the standards under the same chromatographic conditions as reported by Donno et al. [57 (link)]. The standards, purchased from Sigma-Aldrich (Saint Louis, MO, USA), were the following: flavonols (hyperoside, isoquercitrin, quercetin, quercitrin, and rutin), catechins (catechin and epicatechin), benzoic acids (ellagic and gallic acids), cinnamic acids (caffeic, chlorogenic, coumaric, and ferulic acids), and vitamin C (ascorbic and dehydroascorbic acids). All the analyses were performed in three replicates.
Each compound was determined comparing the retention times and UV spectra with the standards under the same chromatographic conditions as reported by Donno et al. [57 (link)]. The standards, purchased from Sigma-Aldrich (Saint Louis, MO, USA), were the following: flavonols (hyperoside, isoquercitrin, quercetin, quercitrin, and rutin), catechins (catechin and epicatechin), benzoic acids (ellagic and gallic acids), cinnamic acids (caffeic, chlorogenic, coumaric, and ferulic acids), and vitamin C (ascorbic and dehydroascorbic acids). All the analyses were performed in three replicates.
6
HPLC-MS/MS Analysis of Tamarixetin
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HPLC-MS/MS analysis was operated using an Agilent 1200 high-performance liquid chromatography coupled with an Aglient 6430 series triple quadrupole mass spectrometer with an electrospray ionization (ESI) source. CORTECS C18 column (4.6 mm × 150 mm, 2.7 μm) was employed for the separation of tamarixetin and IS. The mobile phase components were methanol-water-formic acid (85 : 15 : 0.1, v/v), injected at a flow rate of 0.3 mL/min for isocratic elution. The column temperature and injection volume were optimized at 30°C and 5 μL, respectively. All mass spectrometry data were acquired with Mass Hunter workstation software.
The ESI source was performed in the negative ion mode and finalized as follows: capillary voltage was set at −4000 V for the negative ionization mode; drying gas temperature was 300°C, flow was 11 L/min, and nebulizing gas pressure was 15 psi. The optimal MRM fragmentation transitions were m/z 315.0 ⟶ 151.1 with a fragmentor voltage of 145 V and a collision energy (CE) of 22 V for tamarixetin and m/z 271.1 ⟶ 151.0 with a fragmentor voltage of 153 V and CE of 8 V, for naringenin (IS). The chemical structures and mass spectrum of tamarixetin and naringenin are shown inFigure 1 .
The ESI source was performed in the negative ion mode and finalized as follows: capillary voltage was set at −4000 V for the negative ionization mode; drying gas temperature was 300°C, flow was 11 L/min, and nebulizing gas pressure was 15 psi. The optimal MRM fragmentation transitions were m/z 315.0 ⟶ 151.1 with a fragmentor voltage of 145 V and a collision energy (CE) of 22 V for tamarixetin and m/z 271.1 ⟶ 151.0 with a fragmentor voltage of 153 V and CE of 8 V, for naringenin (IS). The chemical structures and mass spectrum of tamarixetin and naringenin are shown in
7
Multimodal Imaging and Analysis Protocol
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The instruments used were OLYMPUS IMT-2 fluorescence microscope (OLYMPUS, Japan), laser confocal scanning microscopy (LCSM, Thermo scientific, USA), Agilent 1200 high performance liquid chromatography (HPLC, Agilent, USA), ELx800 microplate reader (BioTek, USA), flow cytometry (BD Biosciences, USA), Roche LightCycler 480II real-time PCR (Roche, Switzerland), PowerPac Basic electrophoresis instrument (BIO-RAD, USA).
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