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Bio-Assays for Oxidative Stress Status
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Field samples should be spun down in a hematocrit centrifuge directly after collection in order to store the plasma separately. We recommend transferring the separated plasma to a 0. See Langille et al. A single HPLC system and two workers can expect to process a calibration curve and about plasma samples in one working day. Table 1. Mix well. Prepare an MDA calibration by serially diluting the freshly prepared 1. Prepare fresh calibration curve before every batch of samples.
See Table 2. Table 2. Preparation of serially diluted 1,1,3,3-tetraethoxypropane standards for the MDA quantification method Store standards on ice until use. Prepare standard 1. Note: We prefer to run all in triplicate, so we purchased a label printer which can make the repeated labeling of tubes much easier. Briefly vortex the samples, then incubate in the ice bath for 5 min.
Note: The QC is a pooled sample of all the plasma you will process in one day. Injecting this QC throughout the HPLC run process every samples will allow you to observe if there are any issues with the runs or decomposition of your samples. Subject the vials to HPLC analysis in triplicate within 12 h of sample preparation. FRAP assay Notes: a.
FRAP reagent is light sensitive, and unstable in ambient air. The reagent must be prepared directly before use. Prepare a working iron sulfate solution by diluting 1 ml of the mM stock with 9 ml of distilled water in a 15 ml centrifuge tube. Use this 30 mM solution to prepare the calibration solutions in 15 ml centrifuge tubes see Table 3.
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Table 3. Dilution table of iron sulfate standards for the FRAP assay Obtain a standard flat bottomed well plate, and plan for where the position of the calibration curves and the samples will be positioned. We like to run 2 calibration curves per plate to account for errors. Be sure that the two drops mix well. Incubate the solution for 5 min at room temperature on an orbital shaker at rpm.
After 30 min, the absorbance at nm is read vs. HASC assay Notes: a. Oxidant solution is unstable in air and should be prepared with care. Adjustment of the pH should be carried out in a fume hood as small quantities of chlorine gas will be produced. Prepare the oxidant solution as in the Recipes section. Protect the solution from light by wrapping the bottle in aluminum foil, and work quickly with the solution. From the oxidant solution, prepare a calibration curve of HClO by diluting the 1x to 2x, 4x, 8x, 16x and 32x dilutions.
Prepare 1 ml of each solution. Note: We prefer to run at least two calibration curves per plate to account for any error which may occur. Vortex the plate on a 2D shaker at rpm for 30 s.
Oxidative stress - Wikipedia
Note: Do not add oxidant solution to the standard wells. The standard is directly read as a serial dilution of HClO. Shake the plate on an orbital shaker for 10 min at room temperature and rpm. Incubate the plate on an orbital shaker for 1 min at room temperature and rpm. Read the absorbance of the wells at nm. Export this data and use it to prepare the calibration curves and to quantify samples. Average triplicate data in its raw format then quantify using the standard linear calibration see Langille et al.
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FRAP assay Export the absorbance data of the wells and take the average of the triplicate wells. Use this data to prepare the linear calibration and quantify samples see Langille et al. HASC assay Export the absorbance data of the wells and take the average of the triplicate wells.
Related Bio-Assays for Oxidative Stress Status
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