Fifty-microliters of overnight cultures of S. epidermidis or S. haemolyticus strains were inoculated into 5 ml of tryptic soy broth and aerobically cultured at 37°C for 18 h or 14 h. The culture supernatants were dried using a centrifugal evaporator. The precipitates were dissolved in 40% acetonitrile and the soluble fraction was dried. The precipitates were dissolved in water and analyzed in reversed phase HPLC using SOURCE 5RPC ST 4.6/150 column (GE Healthcare, Tokyo, Japan) and 50% acetonitrile in 0.1% trifluoroacetic acid for 3 min and a water/acetonitrile gradient in 0.1% trifluoroacetic acid from 50% to 90% acetonitrile for 20 min at a flow rate of 1 ml/min (600E, Waters, Milford, MA). Absorbance at 215 nm was measured using a 2998 Photodiode Array Detector (Waters). Each PSM species was identified by liquid chromatography/electrospray-ionization mass spectroscopy (LC 1100 series, Agilent Technologies, Santa Clara, CA; ESI-MS, Bio-TOFQ, Bruker Daltonics, Billerica, MA) and the predicted molecular masses of PSMs [25] (link), [27] . S. epidermidis PSMα and PSMδ were eluted at the same retention time in this assay condition.
Lc 1100 series
Manufactured by Agilent Technologies
Sourced in United States, Singapore, Germany
The Agilent 1100 series is a line of high-performance liquid chromatography (HPLC) systems. The core function of the 1100 series is to provide reliable and accurate separation, identification, and quantification of chemical compounds in liquid samples.
Automatically generated - may contain errors
Lab products found in correlation
6490 triple quadrupole ms, by Agilent Technologies (2 mentions)
2998 photodiode array detector, by Waters Corporation (1 mentions)
Biotof q, by Bruker (1 mentions)
Jet stream technology, by Agilent Technologies (1 mentions)
Luna c5 column, by Phenomenex (1 mentions)
Icp ms 7500a, by Agilent Technologies (1 mentions)
Onyx monolithic c18 column, by Phenomenex (1 mentions)
Vacuum concentrator, by Eppendorf (1 mentions)
Silica gel 60, by Merck Group (1 mentions)
Lc msd tof mass spectrometer, by Agilent Technologies (1 mentions)
Dpx 200 spectrometer, by Bruker (1 mentions)
Picogreen assay, by Thermo Fisher Scientific (1 mentions)
Masshunter workstation, by Agilent Technologies (1 mentions)
30 m db 88 capillary column, by Agilent Technologies (1 mentions)
6890 series gc, by Agilent Technologies (1 mentions)
Pursuit xrs c18 column, by Agilent Technologies (1 mentions)
Trypsin, by Merck Group (1 mentions)
Eclipse xdb c18 analytical column, by Agilent Technologies (1 mentions)
20 protocols using lc 1100 series
1
Purification and Identification of Staphylococcal PSMs
2
Quantification of Resveratrol by LC-MS/MS
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The LC–MS/MS system consists of an Agilent LC 1100 series (Agilent Technologies, CA, USA) binary pump, a vacuum degeneration unit and an auto-sampler system connected to a 6490 triple quadrupole MS equipped with an Agilent jet stream technology electrospray ionization (ESI) source. Chromatographic separation was performed by an analysis Sepax BR-C18 (5 µm, 120 Å 1.0 × 100 mm) column. The maintaining temperature of column was set at 30 °C. The temperature of the auto-sampler was set at 4 °C. Of the sample solution 2 µL was injected and the analytes were eluted under the isocratic condition with acetonitrile and 0.1% formic acid in water (60%:40%, v/v) pumped at a constant flow of 0.10 mL/min. To detection of the resveratrol in the analytes, the MS/MS system was performed under negative ESI and the multiple reactions monitoring (MRM) mode. The MS operational parameters were: Argon as a collision gas; capillary voltage at 5 kV; gas temperature at 225 °C; gas flow 14.1 I/min; nebulizing gas at 40 psi; collision energies of 18 for resveratrol and the precursor to product ion transitions of 184.8–226.9 m/z for resveratrol.
3
LC-MS Metabolomics Profiling of Serum Samples
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Analysis was performed with an Agilent LC (1100 series) coupled to an Agilent high resolution MS (Model 6550 QTOF, Santa Clara, CA, USA) as previously reported [22 (link)]. Briefly, 10 μl of extracts were slowly loaded on to a Luna C5 column (Phenomenex, Los Angeles, CA) with a 22-min gradient elution of mobile phase A (methanol/0.5% acetic acid = 5:95) and mobile phase B (isopropanol/methanol/0.5% acetic acid = 60:35:5). The electrospray was operated in negative electrospray-ionization (ESI) mode. Tandem MS/MS spectra were obtained on the same platform in data-dependent mode (immediately after data collection) or targeted mode (analysis of the selected features). Full LC-MS acquisition parameters were previously published [22 (link)].
Approximately one third of the serum samples had a gelled consistency that resulted from an additive to the cryostraws [22 (link), 44 , 45 ]. Pairs with at least one gelled sample were analyzed in one batch (batch 1, n = 96), and the remaining (non-gelled) pairs were analyzed in a second batch (batch 2, n = 36).
Approximately one third of the serum samples had a gelled consistency that resulted from an additive to the cryostraws [22 (link), 44 , 45 ]. Pairs with at least one gelled sample were analyzed in one batch (batch 1, n = 96), and the remaining (non-gelled) pairs were analyzed in a second batch (batch 2, n = 36).
4
Arsenic Speciation in Algal Samples
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We determined total As and As speciation according to the previous methods used for algal sample preparation and As analysis [9 ]. In brief, approximately 0.02 g of oven-dried algal samples were treated overnight by means of microwave assisted digestion. Afterwards, samples were further diluted to measure As using ICP-MS. We had a good recovery rate (92.3% ± 5.6%) using a standard reference sample (GBW08521, the National Research Center for Standard Materials of China). Additionally, approximately 0.02 g of the freeze-dried algal samples was treated overnight. After microwave assisted digestion, samples were filtered using 0.45 μm filters. We then used HPLC-ICP-MS (Agilent LC1100 series coupled with the Agilent ICP-MS 7500a) to measure As speciation in algal extracts and media.
5
Quantification of Acetaminophen in Metabolite Samples
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300 μL of sample containing metabolites were mixed with 100 μL of 100 ng/mL internal standard APAP-D4 and dried using a concentrator at 30 °C (Eppendorf, Germany) under vacuum. The dried residues were reconstituted using 100 μL methanol containing 0.1% formic acid and centrifuged at 10, 000 rpm for 10 min at 4 °C. 80 μL of supernatant was then transferred to the LC/MS sample vials and used for measurement by LC/MS system (LC: 1100 series, Agilent, Singapore; MS: LCQ Deca XP Max, Finnigan, Singapore) with 100 × 3.0 mm onyx-monolithic C18 column (Phenomenex, USA). The mobile phase consisted of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in methanol) with a flow rate of 0.8 mL/min. The elution scheme for the measurement of acetaminophen involved solvent B that was gradually increased from 6% to 90% over 6 min. The MS parameter settings were as follows: spray voltage 5 kV; sheath gas flow rate: 80; auxiliary gas flow rate: 20; capillary temperature: 350 °C; tube lens: 45 V; and capillary voltage: 30 V.
6
CYP Activity Quantification in Hepatocytes
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For CYPs specific activity analysis, hepatocytes were cultured for 8 or 14days before adding CYP-specific substrates diluted in Krebs-Henseleit buffer (KHB) and incubated for 1.5 hours. Hepatocytes in perfusion cultured were removed from the bioreactor and transferred to multi-well plates for this experiment. The samples were collected and stored at −80 °C until LC-MS measurement. To conduct the LC-MS measurement, 50 µL of 100ng/mL internal standards were added to the samples and the mixture was dried using Techne® Sample Concentrator (Techne, UK). The dried residues were reconstituted using 100 µL of methanol containing 0.1% formic acid. The supernatant was then analysed using LC-MS system (LC: 1100 series, Agilent, US; MS: LCQ Deca XP Max, Thermo Finnigan, US) with 100 × 3.0 mm onyx-monolithic C18 column (Phenomenax, USA) as reported previously74 (link).
7
LC-MS/MS Analysis of Resveratrol and Curcumin
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The LC-MS/MS system consisted of an Agilent LC 1100 series (Agilent Technologies, CA, USA) binary pump, a vacuum degasser, and an auto-sampler system connected to a 6490 triple quadrupole MS equipped with an electrospray ionization (ESI) source from Agilent jet stream technology. Chromatographic separation was achieved by an analytical Sepax BR-C18 (5 μm, 120 Å 1.0×100 mm) column. The column temperature was maintained at 30°C. The temperature of the auto-sampler was set at 4°C. The sample solutions were injected (2 μL), and the analytes were eluted at a constant flow of 0.150 mL/min of the mobile phase (ratio of acetonitrile to 0.1% formic acid in water, 60:40%, v/v). The isocratic separation run time was 5 min. The MS/MS system was run under negative and/or positive ESI and multiple reactions monitoring (MRM) mode was used to identify the compounds of interest. The operational parameters of MS were: argon as a collision gas; capillary voltage at 5 kV; gas temperature at 225°C; gas flow of 15.1 L/min; and collision energies of 18 and 14 eV for resveratrol and curcumin, respectively. Analyte detection was performed in MRM mode to monitor the precursor to product ion transitions of 226.8-184.8 m/z for resveratrol and 367.1-148.9 for curcumin.
8
Cytochrome P450 Enzymatic Activity Assay
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Basal activities
of two important cytochrome P450 (CYP) enzymes (i.e., CYP1A2 and CYP3A4),
were determined. Following differentiation, HepaRG-Hep were incubated
in 100 μL of Krebs–Henseleit buffer (KHB) with a cocktail
of CYPs substrates (Table S6 ) in 96 well
plates for 2 h at 37 °C. Dimethyl sulfoxide (DMSO) was selected
as a solvent to dissolve the CYP substrates as stock solutions. The
stock solutions of CYP substrates were diluted in KHB to a final concentration
such that DMSO did not exceed 0.1%. The metabolites generated by HepaRG-Hep
were then analyzed using liquid chromatography-mass spectrometry (LCMS).
In general, 100 μL of supernatants generated by HepaRG-Hep were
spiked with 100 μL of APAP D4 as internal standards (Table S6 ). The mixtures were dried using a vacuum
concentrator (Eppendorf, Germany) in room temperature for at least
6 h to eliminate the solvent. 100 μL of methanol containing
0.1% formic acid was added into each of the dried residues and followed
by a vortex mixing. After centrifuging at 10 000g for 10 min at 4 °C, 60 μL of the sample supernatant was
collected for LCMS analysis (LC: 1100 series, Agilent, Singapore;
MS:LCQ Deca XP Max, Finnigan, Singapore). The drug metabolite products
are acetaminophen (APAP) and hydroxy midazolam for CYP1A2 and CYP3A4,
respectively.
of two important cytochrome P450 (CYP) enzymes (i.e., CYP1A2 and CYP3A4),
were determined. Following differentiation, HepaRG-Hep were incubated
in 100 μL of Krebs–Henseleit buffer (KHB) with a cocktail
of CYPs substrates (
plates for 2 h at 37 °C. Dimethyl sulfoxide (DMSO) was selected
as a solvent to dissolve the CYP substrates as stock solutions. The
stock solutions of CYP substrates were diluted in KHB to a final concentration
such that DMSO did not exceed 0.1%. The metabolites generated by HepaRG-Hep
were then analyzed using liquid chromatography-mass spectrometry (LCMS).
In general, 100 μL of supernatants generated by HepaRG-Hep were
spiked with 100 μL of APAP D4 as internal standards (
concentrator (Eppendorf, Germany) in room temperature for at least
6 h to eliminate the solvent. 100 μL of methanol containing
0.1% formic acid was added into each of the dried residues and followed
by a vortex mixing. After centrifuging at 10 000g for 10 min at 4 °C, 60 μL of the sample supernatant was
collected for LCMS analysis (LC: 1100 series, Agilent, Singapore;
MS:LCQ Deca XP Max, Finnigan, Singapore). The drug metabolite products
are acetaminophen (APAP) and hydroxy midazolam for CYP1A2 and CYP3A4,
respectively.
9
Boc-Glu(OBzl) Synthesis and Characterization
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All reagents used were commercial
products and were used without further purification unless otherwise
indicated. Boc-Glu(OBzl)–OH (Boc-l -glutamic acid 5-benzyl
ester,15 ) was purchased from Sigma-Aldrich. Flash chromatography
(FC) was performed using silica gel 60 (230–400 mesh, Sigma-Aldrich). 1H NMR spectra were obtained at 200 MHz and 13C
NMR spectra were recorded at 50 MHz (Bruker DPX 200 spectrometer).
Chemical shifts are reported as δ values (parts per million)
relative to remaining protons in deuterated solvent. Coupling constants
are reported in hertz. The multiplicity is defined by s (singlet),
d (doublet), t (triplet), q (quartet), p (pentet), br (broad), or
m (multiplet). HPLC analyses were performed on an Aglient LC 1100
series. High-resolution MS experiments were performed using an Agilent
Technologies LC/MSD TOF mass spectrometer.
products and were used without further purification unless otherwise
indicated. Boc-Glu(OBzl)–OH (Boc-
ester,
(FC) was performed using silica gel 60 (230–400 mesh, Sigma-Aldrich). 1H NMR spectra were obtained at 200 MHz and 13C
NMR spectra were recorded at 50 MHz (Bruker DPX 200 spectrometer).
Chemical shifts are reported as δ values (parts per million)
relative to remaining protons in deuterated solvent. Coupling constants
are reported in hertz. The multiplicity is defined by s (singlet),
d (doublet), t (triplet), q (quartet), p (pentet), br (broad), or
m (multiplet). HPLC analyses were performed on an Aglient LC 1100
series. High-resolution MS experiments were performed using an Agilent
Technologies LC/MSD TOF mass spectrometer.
10
Quantifying Hepatocyte Functionality in Bioreactor
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Perfused culture media were collected daily for each chamber of the bioreactor and kept at −20 °C until the assay was carried out. The control was the supernatant of the static cultured hepatocytes on a 24-well culture plate. Urea production was quantified by BUN urea nitrogen test (Stanbio Laboratory, Boerne, TX, USA). The CYP level was quantified on the CYP specific metabolites, with the internal standards (Cyp1a2, Cyp3a2: 100 ng/mL Acetaminophen-D4; Cyp2b2: 50 ng/mL OHbupropion-D6), by LC/MS measurement (LC: 1100 series, Agilent, Singapore; MS: LCQ Deca XP Max, Finnigan, Singapore). All functional data were normalized to the seeded cell numbers, which were determined by the PicoGreen assay (Molecular Probes, Eugene, OR, USA).
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