Cultures of L. monocytogenes were grown in BHI at 37°C with shaking overnight. Bacteria were then centrifuged and 1 mL of the resulting supernatant was filtered through a 0.2μm-pore-size syringe filter (09-740-113; Fisher Scientific). Supernatant samples were next treated with 2μL of H2SO4 to precipitate any components that might be incompatible with the running buffer. The samples were then centrifuged at 16000 × g for 10 min and then 200μL of each sample transferred to an HPLC vial. HPLC analysis was performed using a ThermoFisher (Waltham, MA) Ultimate 3000 UHPLC system equipped with a UV detector (210 nm). Compounds were separated on a 250 × 4.6 mm Rezex© ROA-Organic acid LC column (Phenomenex Torrance, CA) run with a flow rate of 0.2 mL min−1 and at a column temperature of 50 °C. The samples were held at 4 °C prior to injection. Separation was isocratic with a mobile phase of HPLC grade water acidified with 0.015 N H2SO4 (415 μL L−1). At least two standard sets were run along with each sample set. Standards were 100, 20, 4, and 0.8mM concentrations of lactate or acetate. The resultant data was analyzed using the Thermofisher Chromeleon 7 software package.
Rezex roa organic acid lc column
Manufactured by Phenomenex
Sourced in Canada, Germany
The Rezex© ROA-Organic acid LC column is designed for the analysis of organic acids. It features an ion-exchange resin-based stationary phase that allows for the separation and quantification of various organic acids, including acetic, lactic, and succinic acids, among others. The column is compatible with aqueous mobile phases and provides reliable performance for routine organic acid analysis.
Automatically generated - may contain errors
Lab products found in correlation
Chromeleon 7, by Thermo Fisher Scientific (4 mentions)
Ultimate 3000 uhplc system, by Thermo Fisher Scientific (3 mentions)
Spd 20av uv detector, by Shimadzu (3 mentions)
Hplc system, by Shimadzu (3 mentions)
Agilent 1260 infinity rid, by Agilent Technologies (2 mentions)
Labsolutions software package, by Shimadzu (2 mentions)
Ultimate 3000 uhplc system, by Shimadzu (2 mentions)
8 protocols using rezex roa organic acid lc column
1
Quantification of Bacterial Metabolites by HPLC
2
Analyzing Short-Chain Fatty Acids in Cecal Digesta
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Weekly feed samples were analyzed for dry matter according to standard procedures method 930.15 (AOAC, 2005 ). The concentration of SCFA (lactic, formic, acetic, propionic, isobutyric, and n-butyric) in the ceca digesta was assayed according to Leung et al. (2018) (link). Briefly, the digesta was thawed and approximately 0.1 g was resuspended with 1 mL 0.005N H2SO4 (1:10, wt/vol) in a microcentrifuge tube. The tube was vortexed vigorously until sample was completely dissolved, centrifuged at 11,000 × g for 15 min, 400-µL supernatant transferred into a high-performance liquid chromatography (HPLC) vial, and 400 µL of 0.005N H2SO4 buffer added. The resulting digesta fluid was then assayed for SCFA using HPLC (Hewlett Packard 1100, Germany) with Rezex ROA-Organic Acid LC column, 300 × 7.8 mm from Phenomenex and Refractive Index detector at 40 °C (Agilent 1260 Infinity RID from Agilent Technologies, Germany). Twenty microliters of the resulting sample was injected into the column, with a column temperature of 60 °C and mobile phase of 0.005N H2SO4 buffer at 0.5mL/min isocratic for 35 min. The detector was heated to 40 °C. The plasma urea nitrogen and creatinine were analyzed by photometrics using a Roche Cobas 6000 c501 biochemistry analyzer (Roche Diagnostics USA, Indianapolis, IN) at the Animal Health Laboratory (University of Guelph, Guelph, ON).
3
Analyzing Short-Chain Fatty Acids in Cecal Digesta
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The concentrations of SCFA (lactic, formic, acetic, propionic, isobutyric, and n-butyric) in the ceca digesta were assayed according to Leung et al. (2018 (link)). Briefly, the digesta was thawed and approximately 0.1 g was resuspended with 1 mL of 0.005 N H2SO4 (1:10 wt/vol) in a microcentrifuge tube. The tube was vortexed vigorously until the sample was completely dissolved, centrifuged at 11,000 x g for 15 min, and 400 μL of the supernatant was transferred into a high-pressure liquid chromatography vial and 400 μL of 0.005 N H2SO4 buffer was added. The resulting digesta fluid was then assayed for SCFA using high-pressure liquid chromatography (Hewlett Packard 1100, Germany) with a Rezex ROA-Organic Acid LC column, 300 × 7.8 mm from Phenomenex, and a refractive index detector at 40°C (Agilent 1260 Infinity RID; Agilent Technologies, Germany). Twenty microliter of the resulting sample was injected into the column, with a column temperature of 60°C and mobile phase of 0.005 N H2SO4 buffer at 0.5 mL/min isocratic for 35 min. The detector was heated to 40°C.
4
HPLC Analysis of Organic Acids
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Supernatant samples (200uL) were thawed in a room temperature water bath before addition of 2 μL of H2SO4 to precipitate any components that might be incompatible with the running buffer. The samples were then centrifuged at 3500 × rpm for 10 min and then 150 μL of each sample was filtered through a 0.2 μm filter using a vacuum manifold before transferring 70 μL of each sample to an HPLC vial. HPLC analysis was performed using a Shimadzu HPLC system equipped with a SPD-20AV UV detector (210 nm). Compounds were separated on a 250 × 4.6 mm Rezex© ROA-Organic acid LC column (Phenomenex Torrance, CA) run with a flow rate of 0.2 mL min−1 and at a column temperature of 50 °C. The samples were held at 4 °C prior to injection. Separation was isocratic with a mobile phase of HPLC grade water acidified with 0.015 N H2SO4 (415 μL L−1). Two standard sets were run along with each sample set, one before all samples and one after all samples. Standards were 100, 20, and 4 mM concentrations of butyrate, succinate, lactate, and acetate, respectively. The injection volume for both sample and standard were 25 μL. The resultant data was analyzed using Shimadzu LabSolutions software package with peaks manually reintegrated if necessary.
5
Quantification of Bacterial Butyrate Production
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Butyrate was quantified in bacterial culture supernatants as previously described (50 (link)). Overnight cultures of C. difficile 630 grown in mRCM and sub-cultured into mRCM with 50 mM of butyrate and NaCl bacterial cultures. At the time points specified in the figures, cultures were centrifuged at 3,000 × g for 5 min and the resulting supernatant was collected, 0.22 µm filtered, and stored −20˚C. Supernatants were thawed and H2SO4 was added to a final concentration of 18 mM. Samples were mixed, incubated 2 min at room temperature and centrifuged at 21,000 × g for 10 min at 4°C. Soluble fractions were aliquoted into HPLC vials. In addition, 100, 20, and 4 butyrate standards were prepared in mRCM or BDMM, as appliable and processed as above. HPLC analysis was performed with a ThermoFisher (Waltham, MA) Ultimate 3000 UHPLC system equipped with a UV detector (210 nm). Compounds were separated on a 250 × 4.6 mm Rezex ROA-Organic acid LC column (Phenomenex Torrance, CA) run with a flow rate of 0.3 mL min−1 and at a column temperature of 50°C. Separation was isocratic with a mobile phase of HPLC grade water acidified with 0.015 N H2SO4. Resulting data were analyzed with ThermoFisher Chromeleon 7 and butyrate concentrations in culture supernatants were determined by analysis against a standard curve as described above.
6
Quantification of Butyrate in C. difficile
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Butyrate was quantified in bacterial culture supernatants as previously described [43 (link)]. Overnight cultures of C. difficile 630 grown in mRCM and sub-cultured into mRCM with 50 mM of butyrate and NaCl bacterial cultures. At the time points specified in the figures, cultures were centrifuged at 3000 x g for 5 minutes and the resulting supernatant was collected, 0.22µm filtered, and stored −20 °C. Supernatants were thawed and H2SO4 was added to a final concentration of 18 mM. Samples were mixed, incubated 2 minutes at room temperature and centrifuged at 21,000 x g for 10 minutes at 4°C. Soluble fractions were aliquoted into HPLC vials. In addition, 100 mM, 20 mM, and 4 mM butyrate standards were prepared in mRCM or BDMM, as appliable and processed as above. HPLC analysis was performed with a ThermoFisher (Waltham, MA) Ultimate 3000 UHPLC system equipped with a UV detector (210 nm). Compounds were separated on a 250 × 4.6 mm Rezex© ROA-Organic acid LC column (Phenomenex Torrance, CA) run with a flow rate of 0.3 mL min−1 and at a column temperature of 50 °C. Separation was isocratic with a mobile phase of HPLC grade water acidified with 0.015 N H2SO4. Resulting data were analyzed with ThermoFisher Chromeleon 7 and butyrate concentrations in culture supernatants were determined by analysis against a standard curve as described above.
7
HPLC Analysis of Organic Acids
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Supernatant samples were thawed in a room temperature water bath before addition of 2 μL of H2SO4 to precipitate any components that might be incompatible with the running buffer. The samples were then centrifuged at 2400 × g for 10 min and then 150 μL of each sample was filtered through a 0.2 μm filter using a vacuum manifold before transferring 70 μL of each sample to an HPLC vial. HPLC analysis was performed using either a ThermoFisher (Waltham, MA) Ultimate 3000 UHPLC system equipped with a UV detector (210 nm) or a Shimadzu HPLC system equipped with a SPD-20AV UV detector (210 nm). Compounds were separated on a 250 × 4.6 mm Rezex© ROA-Organic acid LC column (Phenomenex Torrance, CA) run with a flow rate of 0.2 mL min−1 and at a column temperature of 50 °C. The samples were held at 4 °C prior to injection. Separation was isocratic with a mobile phase of HPLC grade water acidified with 0.015 N H2SO4 (415 µL L−1). At least two standard sets were run along with each sample set. Standards were 100, 20, and 4 mM concentrations of butyrate, succinate, lactate, and acetate, respectively. For most runs, the injection volume for both sample and standard was 25 µL. The resultant data was analyzed using the Thermofisher Chromeleon 7 software package or Shimadzu LabSolutions software package.
8
HPLC Analysis of Organic Acids
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Supernatant samples were thawed in a room temperature water bath before addition of 2 𝜇L of H2SO4 to precipitate any components that might be incompatible with the running buffer. The samples were then centrifuged at 2400xg for 10 minutes and then 150 𝜇L of each sample was filtered through a 0.2 𝜇m filter using a vacuum manifold before transferring 70 𝜇L of each sample to an HPLC vial. HPLC analysis was performed using either a ThermoFisher (Waltham, MA) Ultimate 3000 UHPLC system equipped with a UV detector (210 nm) or a Shimadzu HPLC system equipped with a SPD-20AV UV detector (210 nm). Compounds were separated on a 250 x 4.6 mm Rezex© ROA-Organic acid LC column (Phenomenex Torrance, CA) run with a flow rate of 0.2 ml min -1 and at a column temperature of 50ºC. The samples were held at 4ºC prior to injection.
Separation was isocratic with a mobile phase of HPLC grade water acidified with 0.015 N H2SO4
(415 µL L -1 ). At least two standard sets were run along with each sample set. Standards were 100, 20, and 4 mM concentrations of butyrate, succinate, lactate, and acetate, respectively. For most runs, the injection volume for both sample and standard was 25 µl. The resultant data was analyzed using the Thermofisher Chromeleon 7 software package.
Separation was isocratic with a mobile phase of HPLC grade water acidified with 0.015 N H2SO4
(415 µL L -1 ). At least two standard sets were run along with each sample set. Standards were 100, 20, and 4 mM concentrations of butyrate, succinate, lactate, and acetate, respectively. For most runs, the injection volume for both sample and standard was 25 µl. The resultant data was analyzed using the Thermofisher Chromeleon 7 software package.
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