Data processing was performed in the TargetLynx application manager of MassLynx 4.1 (Waters). Absolute quantification was performed by interpolation from calibration curves prepared by serial dilutions of analytical grade standards (Sigma-Aldrich). Calibration curves were calculated by least-squares regression with 1/x weighting, and all response factors of both standards and biological extracts were corrected by the response factor of the corresponding U13C(15N)-isotopologue. Extract concentrations were corrected for dilutions and concentrations performed during sample preparation, and normalized to measured cell density (cells/L) or total DW (g/L) in cell lines and microorganisms, respectively. Intracellular metabolite concentrations were calculated from experimental cell volumes (pL) of cell lines and estimated using literature values for specific cell volume (L/g) of microorganisms (Table 2 ).
Analytical grade standards
Manufactured by Merck Group
Sourced in Australia
Analytical grade standards are a type of reference material used in analytical chemistry to ensure the accuracy and precision of analytical measurements. These standards are highly purified, well-characterized chemical substances with known concentrations or properties, which are used to calibrate and validate analytical instruments and methods.
Automatically generated - may contain errors
4 protocols using analytical grade standards
1
Absolute Quantification of Metabolites
2
Cellular Redox State Quantification
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To estimate the changes in global cellular redox state, the ratio of oxidized/total glutathione (GSSG/total GSH) was determined from cell lysates by capillary electrophoresis (Agilent 7100, Agilent Technologies, Santa Clara, CA, USA) equipped with a polyimide-coated fused silica capillary (68 cm × 50 μm). Cells were gently scraped and collected in centrifugation tube with its culture medium. Tube was centrifuged at 300 ×g for 5 minutes; cell pellets were quickly washed with ice-cold phosphate-buffered saline and immediately lysed in acetonitrile/water (3 : 2, v/v). Lysates were centrifuged at 20,000 ×g for 20 min at 4°C. Supernatant containing glutathione was collected, snap frozen, and stored in liquid nitrogen up to 6 hours before the analysis. Separation was done in 40 mM phosphate buffer pH 7.0, voltage was set at 30 kV, and absorbance was measured at 195 nm. Reduced and oxidized glutathione were detected as separate peaks and identified by a comparison with analytical grade standards (Sigma-Aldrich). GSSG percentage was calculated from the areas of the individual peaks of GSH and GSSG as described previously [26 (link)].
3
Quantitative Metabolite Profiling in Cell Lines
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Downstream data processing was performed in TargetLynx application manager of MassLynx 4.1 (Waters). Ion abundances were normalized to total ion abundance in DU145 and HEK293 extracts. Metabolite levels in HAP1, JJN3, RPMI 8226, MC/CAR, HL60, NB4, and primary monocyte extracts were absolutely quantified by interpolation from calibration curves prepared by serial dilutions of analytical grade standards (Sigma-Aldrich) calculated by least squares regression. Response factors of the analytical standards and biological extracts were corrected by the response factor of their corresponding U13C-labeled isotopologue spiked into the samples. Extract concentrations were then normalized to experimental cell density at the time of sampling, measured on a MoxiZ cell counter with type S cassettes (Orflo Technologies).
4
GC-MS/QToF Analysis of Volatile Compounds
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Separation of volatile compounds was carried out using GC–MS/QToF (7890A GC; 7200 QToF; Agilent Technologies, CA, USA). Loaded SPME fibers were desorbed in the injection port at 250 °C for 1 min, and subsequent separation was achieved with an HP-5MS capillary column (30 m × 0.25 mm I.D., 0.25 μm film thickness: Agilent Technologies, CA, USA) using helium as the carrier gas at a flow rate of 1.5 mL min−1. The oven temperature was maintained at 40 °C for 2 min and then programmed to rise from 40 to 230 °C at a rate of 4 °C min−1 and then from 230 to 260 °C at a rate of 10 °C min−1. Mass spectra were recorded after exposing the effluent from the GC to electron ionization (EI) at 70 eV; the mass range collected was 50 to 500 m/z.
Volatiles were identified by comparing their mass spectra to reference compounds in the National Institute of Standards and Technology (NIST) mass spectrometry library and authentic standards where available. Analytical grade standards were purchased from Sigma-Aldrich, Australia. Laboratory-grade deionized water was used to dilute standards, and solutions were heated at 100 °C when needed to dissolve solid compounds. The consistency of the performance of the SPME fiber and GC–MS system was assessed by periodically injecting a test mix of p-cresol, phenol, and toluene at 10 ppm.
Volatiles were identified by comparing their mass spectra to reference compounds in the National Institute of Standards and Technology (NIST) mass spectrometry library and authentic standards where available. Analytical grade standards were purchased from Sigma-Aldrich, Australia. Laboratory-grade deionized water was used to dilute standards, and solutions were heated at 100 °C when needed to dissolve solid compounds. The consistency of the performance of the SPME fiber and GC–MS system was assessed by periodically injecting a test mix of p-cresol, phenol, and toluene at 10 ppm.
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