Endothelial cells in culture or from the lesser curvature of aortas from wild type and CSEiEC mice were incubated with dihydroethidium (DHE, 10 μmol/L) for 20 min in the dark, before being snap frozen in liquid nitrogen. LC-MS/MS of DHE derivatives was performed on a 1290 Infinity UHPLC system (Agilent, Waldbronn, Germany) coupled to a 5500 QTrap triple quadrupole mass spectrometer with a TurboV electro spray ionization source (Sciex Deutschland GmbH, Darmstadt, Germany). DHE and its oxidation products were separated on a C18 Phenomenex Kinetex column (150 × 2.1 mm, 2.6 μm), protected by a Phenomenex C18 guard cartridge, using an acetonitrile/water gradient with 0.1% formic acid. The ion source parameters were set as follows: x-axis and y-axis of the source were set to 5.0 mm, TEM = 300 °C, IS = 3500 V, GS1 = 50 p.s.i., GS2 = 40 p.s.i., CUR = 30 p.s.i., collisionally activated dissociation gas (CAD) = medium. Detection of DHE oxidation products (O2•- and ethidium) was achieved by multiple reaction monitoring (MRM) in positive ion mode. The method allowed the detection of DHE degradation product ethidium and the O2•--specific product. System control and analytical data analysis were processed by Analyst software 1.6.2 and MultiQuant 3.0, respectively.
C18 guard cartridge
Manufactured by Phenomenex
Sourced in Canada
The C18 guard cartridge is a column used in high-performance liquid chromatography (HPLC) and ultra-high-performance liquid chromatography (UHPLC) systems. It is designed to protect the analytical column from contaminants and particulates in the sample, helping to extend the column's lifetime and maintain consistent performance.
Automatically generated - may contain errors
Lab products found in correlation
Prominence liquid chromatography platform, by Shimadzu (3 mentions)
Premier xl triple quadrupole electrospray mass spectrometer, by Waters Corporation (2 mentions)
Synergi 4μ hydro rp 80a column, by Phenomenex (2 mentions)
1290 infinity uhplc system, by Agilent Technologies (1 mentions)
Kinetex column, by Phenomenex (1 mentions)
Shim pack vp ods column, by Shimadzu (1 mentions)
Hplc system, by Shimadzu (1 mentions)
Zorbax eclipse xdb c18 column, by Agilent Technologies (1 mentions)
Reversed phase hplc system, by Jasco (1 mentions)
Pu 2089, by Jasco (1 mentions)
Lc 20at pump, by Shimadzu (1 mentions)
Cto 20a column oven, by Shimadzu (1 mentions)
Dgu 20a5r degasser, by Shimadzu (1 mentions)
C18 hplc column, by Phenomenex (1 mentions)
11 protocols using c18 guard cartridge
1
Quantifying Oxidative Stress in Endothelial Cells
2
Quantification of Farnesol in Cultures
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For quantification of FOH, 3 ml n-hexane/ethanol (9:1, v/v) were added to 1 ml sterile-filtered (0.45 μm) culture supernatant, and the derivatization of farnesol with 9-anthroylnitrile was performed as described previously [20 (link)]. The farnesol extracts were supplemented sequentially with 250 μl 0.08% 9-anthroylnitrile ethylacetate solution, 10 μl 0.2% 1-butanol ethylacetate solution as an internal standard, and 250 μl 0.2% quinuclidine ethylacetate solution. Sample analysis was performed using a Shim-Pack Vp-ODS column (5 μm, 150 × 4.6 mm; Shimadzu) equipped with a C18 guard cartridge (4.0 × 3.0 mm; Phenomenex) on a Shimadzu HPLC system (Kyoto, Japan). A mixture of acetonitrile and water (87:13, v/v) was used as the mobile phase at a flow rate of 1.5 ml/min. The farnesol derivative was detected using a fluorescence detector at an excitation wavelength of 363 nm and emission wavelength of 470 nm. Standard concentrations ranged from 0.1 to 2.0 μM. A weighted 1/concentration linear regression was used to obtain calibration curves from the standards. The regression equations of the calibration curves were used to calculate the concentrations of the samples. FOH results obtained from each culture were also normalized on a metabolic basis or a per-weight basis by dividing the values (μM) by the biofilm results determined by XTT reduction or DW assays, respectively.
3
C-di-GMP Extraction and Quantification from Bacteria
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
C-di-GMP was extracted from solid grown or liquid grown E. coli and P. aeruginosa, as previously described (8 (link)), using 2-chloro AMP as an internal standard. C-di-GMP was extracted from pelleted cells by incubating with 70% perchloric acid on ice for 1 h. The supernatant was retained and neutralized using potassium bicarbonate. Liquid chromatography MS/MS measurements were performed using an Acuity UPLC with a Synergi 4μ Hydro RP 80A column and a C18 Guard Cartridge (Phenomenex) on a Premier XL triple-quadrupole electrospray mass spectrometer (Waters). The m/z 691 > 152 transition was used for c-di-GMP and 382 > 170 for 2-chloro-AMP. The cone voltages and collision energies were 40 V/30 eV and 35 V/20 eV, respectively.
4
Synthesis and Purification of Amino Acid Derivatives
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Two equivalents
of the amino acid, dissolved in 1 mL of water, were added to 1 equiv
of the corresponding PSD, dissolved in 2.5 mL of MeCN. The mixture
was stirred for 3 h at room temperature under light exclusion and
evaporated under vacuum. For purification of the reaction products,
the mixture was redissolved in 3 mL of MeCN and applied for semipreparative
LC-UV on a ZORBAX Eclipse XDB-C18 column (9.4 mm × 250 mm, 5
μm, Agilent, Waldbronn, Germany) equipped with a C18 guard cartridge
(4 mm × 3 mm, Phenomenex, Aschaffenburg, Germany) with a MeCN/H2O gradient applying a binary gradient consisting of water
(solventA ) and MeCN (solvent B ) at a consistent
flow rate of 5 mL/min and a 40 °C column oven temperature. For
purification of agmatine derivatives, both eluents contained additionally
0.1% of FA. The gradient programs are shown in the Supporting Information
inTable S2 . The fractions containing the
products were combined and evaporated under vacuum. All semisynthesized
compounds were structurally characterized according to the descriptions
above.
of the amino acid, dissolved in 1 mL of water, were added to 1 equiv
of the corresponding PSD, dissolved in 2.5 mL of MeCN. The mixture
was stirred for 3 h at room temperature under light exclusion and
evaporated under vacuum. For purification of the reaction products,
the mixture was redissolved in 3 mL of MeCN and applied for semipreparative
LC-UV on a ZORBAX Eclipse XDB-C18 column (9.4 mm × 250 mm, 5
μm, Agilent, Waldbronn, Germany) equipped with a C18 guard cartridge
(4 mm × 3 mm, Phenomenex, Aschaffenburg, Germany) with a MeCN/H2O gradient applying a binary gradient consisting of water
(solvent
flow rate of 5 mL/min and a 40 °C column oven temperature. For
purification of agmatine derivatives, both eluents contained additionally
0.1% of FA. The gradient programs are shown in the Supporting Information
in
products were combined and evaporated under vacuum. All semisynthesized
compounds were structurally characterized according to the descriptions
above.
5
Quantification of c-di-GMP in S. Typhimurium
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
C-di-GMP was extracted from S. Typhimurium grown in 96-well plates using a method adapted from (Hickman and Harwood, 2008 (link); Irie et al., 2012 (link)). Briefly, all experiments were performed on independent cultures in biological triplicates. C-di-GMP was extracted by addition of 9 µl of 70% perchloric acid to each well of the 96 well plate and incubation on ice for 1 hour. After an hour, attached cells were dislodged by pipetting and the supernatant was collected and neutralized using potassium bicarbonate. 2-chloro AMP was used as an internal standard. Liquid chromatography MS/MS measurements were performed using an Acuity UPLC with a Synergi 4μ Hydro RP 80A column and a C18 Guard Cartridge (Phenomenex) on a Premier XL triple-quadrupole electrospray mass spectrometer (Waters). The m/z 691 > 152 transition was used for c-di-GMP and 382 > 170 for 2-chloro-AMP. The cone voltages and collision energies were 40 V/30 eV and 35 V/20 eV, respectively. Thirty microliters of each sample was injected and the ratio of area under the curve of the c-di-GMP channel signal (retention time= 1.6 minutes) was divided by the area under the curve of the 2-chloroAMP signal (retention time= 2.1 minutes). A standard curve of 0 nM to 100 nM c-di-GMP containing 2-chloroAMP was used to quantify c-di-GMP for all samples. C-di-GMP concentration was normalized to total protein concentration.
6
HPLC Analysis of ST Drug in Plasma
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The analysis was carried out on a reversed-phase column (Chromatopak peerless basic C18 column of 250 × 4.6 mm, 5 µm), protected with a Phenomenex C18 guard cartridge with the dimension of 250 × 4.6 mm, 5 µm at room temperature. The column was coupled with a Jasco reversed-phase HPLC system (autosampler and LC-Net II/ADC controller, pump PU-2089) coupled with a UV (Jasco UV-2075 plus) detector, and Jasco-Borwin version 15 was used. The mobile phase ACN:Water:Formic Acid (70:29.5:0.5 v/v) was filtered using a 0.25 µm membrane filter and degassed using ultrasonication. After sonication, at the flow rate of 1 mL/min, the mobile phase was pumped to the system, and the detection wavelength was 280 nm with 30 min run time (Table 1 and Fig. 2).
The stock solution of the drug was prepared by accurately dissolving 1 mg of ST into acetonitrile (1,000 µL). The stock solution was safely stored and protected from light, and further dilutions were prepared. An aliquot (100 µL) for each diluted solution was spiked with 100 µL of blank plasma, yielding concentrations ranging from 5 to 50 µg/mL, to obtain the calibration curve. Quality control (QC) samples were also prepared by following the same procedure mentioned earlier. All solutions were stored for 1 month at a temperature of 4°C.
The stock solution of the drug was prepared by accurately dissolving 1 mg of ST into acetonitrile (1,000 µL). The stock solution was safely stored and protected from light, and further dilutions were prepared. An aliquot (100 µL) for each diluted solution was spiked with 100 µL of blank plasma, yielding concentrations ranging from 5 to 50 µg/mL, to obtain the calibration curve. Quality control (QC) samples were also prepared by following the same procedure mentioned earlier. All solutions were stored for 1 month at a temperature of 4°C.
7
HPLC Profiling of Myrtle Wood Polyphenol Extracts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MWP extracts were analyzed using a Shimadzu prominence liquid chromatography platform (Kyoto, Japan) equipped with two LC-20AT pumps, CTO-20A column oven, DGU-20A5R degasser, SIL-20A autosampler, and SPD20AD detector. Chromatographic separation was conducted on a AichromBond-AQ C18 column (250 mm × 4.6 mm, 5 μm; Abel Industries Ltd., Canada), protected by a Phenomenex® C18 guard cartridge (3 × 4 mm, 5 μm; Torrance, CA, USA). The mobile phase consisted of ACN (A) and 0.1% aqueous formic acid (B) and was delivered at 1.0 mL·min−1 with the following gradient program: 0–25 min, 0–3% B; 25–70 min, 3–22% B; 70–85 min, 22–35% B; 85–95 min, 35-35% B; 95–100 min, 35–100% B; 100–105 min, 100-100% B. The column was maintained at 40°C. At the end of each run, the delivery of 100% A was performed for another 14 min for system reequilibration. The monitor wavelength was set at 235 nm, 254 nm, and 280 nm, respectively. Figure 1 and Table 2 show the HPLC fingerprint and the extracts of MWP.
8
Chromatographic Separation of Compounds
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chromatographic separations were carried out on a Phenomenex C18 analytical column (150 mm × 4.6 mm i.d., 5 μm) connected with a Phenomenex C18 guard cartridge (4 mm × 3 mm i.d., 5 μm). The mobile phase consisted of MeCN—potassium dihydrogen phosphate buffer (20 mM, pH 3.3)—and triethylamine. Wavelength of 270 nm was selected for detection. The injection volume of the sample was 20 μL. The HPLC system was used in an air-conditioned laboratory atmosphere.
9
BSHX Compound Analysis via HPLC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Referring to the study of our research group [21 (link)], BSHX extracts were analyzed using a Shimadzu prominence liquid chromatography platform (Kyoto, Japan) equipped with two LC-20AT pumps, a CTO-20A column oven, a DGU-20A5R degasser, an SIL-20A autosampler, and an SPD20AD detector. Chromatographic separation was conducted on a AichromBond-AQ C18 column (250 mm × 4.6 mm, 5 μm; Abel Industries Ltd., Canada), protected by a Phenomenex® C18 guard cartridge (3 × 4 mm, 5 μm; Torrance, CA, USA). The mobile phase consisted of ACN (A) and 0.1% aqueous formic acid (B) and was delivered at 1.0 mL/min with the following gradient program: 0–35 min, 0–3% B; 35–100 min, 3–22% B; 100–115 min, 22–35% B; 115–130 min, 35–35% B; 130–140 min, 35–100% B; 140–145 min, 40–100% B; and 145–155 min, 100–100% B. The column was maintained at 40°C. At the end of each run, the delivery of 100% A was performed for another 14 min for system reequilibration. The monitor wavelength was set at 235 nm, 254 nm, and 280 nm, respectively.
10
HPLC Analysis of BSHX Compounds
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BSHX extracts were analyzed using a Shimadzu prominence liquid chromatography platform (Kyoto, Japan) equipped with two LC-20AT pumps, CTO-20A column oven, DGU-20A5R degasser, SIL-20A autosampler, and SPD20AD detector. Chromatographic separation was conducted on a AichromBond-AQ C18 column (250 mm × 4.6 mm, 5 μm; Abel Industries Ltd., Canada), protected by a Phenomenex® C18 guard cartridge (3 × 4 mm, 5 μm; Torrance, CA, USA). The mobile phase consisted of ACN (A) and 0.1% aqueous formic acid (B) and was delivered at 1.0 mL/min with the following gradient program: 0–35 min, 0–3%B; 35–100 min, 3–22% B; 100–115 min, 22–35%B; 115–130 min, 35–35%B; 130–140 min, 35–100%B; 140–145 min, 40–100%B; and 145–155 min, 100–100%B. The column was maintained at 40°C. At the end of each run, the delivery of 100% A was performed for another 14 min for system reequilibration. The monitor wavelength was set at 235 nm, 254 nm, and 280 nm, respectively.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!