Name: evaluation of oxidative stress in biological samples using the thiobarbituric acid reactive substances assay
Uploaded: 2020-05-12T12:30:00.000+00:00
Duration: 6 min 19 s
Description: Watch this Scientific Journal Video about Evaluation of Oxidative Stress in Biological Samples Using the Thiobarbituric Acid Reactive Substances Assay at JoVE.com

The research reported here was supported in part by the National Cancer Institute of the National Institutes of Health under award no. R33 CA217702 and the Initiative for Maximizing Student Development (IMSD) program. The content is solely the responsibility of the authors and does not necessarily represent the official view of the National Institutes of Health.

Human serum specimens were obtained from consenting volunteers under IRB approval and according to the principles expressed in the Declaration of Helsinki. Specimens were coded and de-identified before transfer to the analytical laboratory.
1. Sample preparation
<ol>
	<li>HepG2 cell lysates
	<ol>
		<li>Seed about 10 x 106 HepG2 cells per flask in 16 T75 flasks with 14 mL of EMEM media supplemented with 10% fetal bovine serum (FBS) and grow cells for 2 days.</li>
		<li>Prepare RIPA buffer: in a 50 mL tube, add 1.5 mL of 5 M NaCl, 2.5 mL of 1 M Tris-HCl (pH = 7), 500 &#956;L of NP-40 reagent, then bring the final volume to 50 mL with DI water.</li>
		<li>Prepare lysis buffer: aliquot 20 mL of RIPA buffer into a 50 mL tube and add 200 &#956;L of a 100x protease inhibitor solution to inhibit protein and lipid degradation. Store at 4 &#176;C. 
		NOTE: Lysis buffer is compatible with TBARS reagents and does not interfere with absorbance at 532 nm. If planning to use a different lysis buffer or add additional ingredients to the lysis buffer, preliminary validation studies need to be done to verify that lysis buffer components are compatible with the TBARS assay.</li>
		<li>Remove media containing 10% FBS and wash cells 2x with 5 mL of cold, sterile 1x PBS.</li>
		<li>Add 1 mL of lysis buffer to the T75 flasks containing the cells and incubate them for 10 min at room temperature (RT) with constant swirling to ensure the buffer is well-distributed.</li>
		<li>Collect lysates into appropriately labeled 2 mL snap-cap polypropylene tubes and incubate on ice for 10 min.</li>
		<li>Spin the lysates at 5,000 x g for 10 min at RT to collect cell debris, and aspirate supernatants into a single 15 mL tube.</li>
		<li>Concentrate cell lysate supernatant four-fold using a Speed Vac at 50 &#176;C and 3 mbar and make aliquots of 94 &#956;L each into 2 mL snap-cap polypropylene tubes. Store samples at -80 &#176;C until they are used for in vitro oxidation and/or TBARS assay. 
		NOTE: To avoid concentrating the cell lysate&#160;supernatant, cells can also be detached using 3 mL of 1x trypsin, neutralized with 6 mL of media, and washed 2x with 5 mL of cold PBS. Cell pellets can then be reconstituted in 250 &#956;L of lysis buffer, and steps 1.1.6 and 1.1.7 can then be performed.</li>
		<li>Prepare a 35 mM CuCl2 stock solution in acetic acid (pH = 4).
		<ol>
			<li>Prepare acetic acid solution (pH = 4): Dilute 1 &#956;L of glacial acetic acid in 100 mL of DI water (pH should be approximately 4 but confirm this with a pH meter). Add more water or acetic acid to adjust the pH to 4.</li>
			<li>Weight out about 0.1936 g of copper II chloride and dissolve in 10 mL of the acetic acid solution (pH = 4) to make a 144 mM CuCl2 stock. Aliquot 490 &#956;L from this solution and add to 1,510 &#956;L of acetic acid (pH = 4) to make a 35 mM CuCl2 solution.</li>
		</ol>
		</li>
		<li>Aliquot 6 &#956;L from the 35 mM CuCl2 stock solution and add it to six samples containing 94 &#956;L of cell lysate to make a final CuCl2 concentration of about 2 mM. Add 6 &#956;L of an acetic acid solution (pH = 4) that does not have CuCl2 to six samples containing 94 &#956;L of cell lysates to use as controls. The final volume of cell lysate should be 100 &#956;L, which is what will be used for the TBARS assay. 
		NOTE: Making the 35 mM CuCl2 stock solution in acetic acid (pH = 4) is necessary to prevent precipitation of copper hydroxide.</li>
		<li>Incubate samples in an oven at 37 &#176;C for 24 h and perform a TBARS assay on each sample containing a final volume of 100 &#956;L.</li>
		<li>Repeat steps 1.1.9 and 1.1.11 2x on separate days to check the reproducibility of the TBARS assay for HepG2 cell lysates.</li>
	</ol>
	</li>
	<li>Low density lipoproteins 
	NOTE: Typically, pre-purified low density lipoprotein (LDL) contains some amount of EDTA. LDL samples used here contain 0.01% EDTA. EDTA can inhibit the in vitro Cu(II)-mediated oxidation of LDL. Hence, it may be necessary to remove EDTA from LDL samples prior to experiments or analysis. Steps 1.2.1&#8211;1.2.5 describe this process. 
	CAUTION: Sodium hydroxide is corrosive and causes irritation in skin and eyes. Use proper personal protective equipment.
	<ol>
		<li>Aliquot 24 &#956;L from a 5.51 mg/mL LDL stock (protein concentration determined by modified Lowry method using BSA as a standard) into appropriately labeled 1 mL snap-cap polypropylene tubes. Make as many aliquots as needed and store at 4 &#176;C until use in oxidation and/or TBARS assay.</li>
		<li>Prepare a 10 mM HEPES buffer in 0.15 M NaCl adjusted to pH = 7 with NaOH beads: dissolve 4.39 g of NaCl in 0.49 L of water, then add 1.19 g of HEPES. Dissolve well with a stir bar. Add sodium hydroxide beads until pH is 7. Dilute to 0.5 L with water. Store buffer at 4 &#176;C and use within 3 months.</li>
		<li>Add 476 &#956;L of the 10 mM HEPES buffer in 0.15 M NaCl (pH = 7) to the aliquoted LDL samples to bring final volume to 500 &#956;L. Add diluted LDL sample to a 0.5 mL centrifugal spin filter device with a 100K molecular weight cutoff.</li>
		<li>Spin samples at 14,000 x g for 10 min at RT, leaving a final retentate volume of about 30 &#956;L. Reconstitute samples in 480 &#956;L of the 10 mM HEPES buffer in 0.15 M NaCl (pH = 7) and spin again at 14,000 x g for 10 min at RT. Perform this step 2x for a total of four spin-throughs.</li>
		<li>Place filter device upside down into a new 2 mL snap-cap polypropylene tube, and centrifuge at 1000 x g for 2 min to collect LDL sample (final volume = about 30 &#956;L).</li>
		<li>Aliquot sample into appropriately labeled 1 mL tube and add 20 &#956;L of water to each sample to achieve a final volume of 50 &#956;L.</li>
		<li>Preparation of 200 &#956;M CuCl2 stock solution in acetic acid (pH = 4)
		<ol>
			<li>Prepare acetic acid solution (pH = 4): see step 1.1.9.1.</li>
			<li>Prepare a 144 mM CuCl2 stock solution (see step 1.1.9.2), then aliquot 5.5 &#956;L from the 144 mM CuCl2 stock and dissolve in a final volume of 4 mL of acetic acid (pH = 4) to make the 200 &#956;M solution.</li>
		</ol>
		</li>
		<li>Aliquot 2.7 &#956;L from the 200 &#956;M CuCl2 stock solution and add it to six samples containing 50 &#956;L of LDL to achieve a final CuCl2 concentration of ~10 &#956;M. Add 2.7 &#956;L from an acetic acid solution (pH = 4) that does not contain CuCl2 to six samples containing 50 &#956;L of LDL to be used for the controls.</li>
		<li>Incubate LDL samples for 2 h in an oven at 37 &#176;C. After 2 h, bring the final volume to 100 &#956;L for each sample with 10 mM HEPES buffer in 0.15 M NaCl (pH = 7). Immediately perform a TBARS assay.</li>
		<li>Repeat steps 1.2.3&#8211;1.2.9 2x on two different days to test reproductivity of the TBARS assay.</li>
	</ol>
	</li>
	<li>Human serum
	<ol>
		<li>From a human serum sample, make aliquots of 94 &#956;L each into 2 mL snap-cap polypropylene tubes and store samples at -80 &#176;C.</li>
		<li>Prepare a 35 mM CuCl2 stock solution in acetic acid (pH = 4): see step 1.1.9.</li>
		<li>Aliquot 6 &#956;L from the CuCl2 stock solution and add it to six samples containing 94 &#956;L of human serum to make a final CuCl2 concentration of about 2 mM. Add 6 &#956;L of an acetic acid solution (pH = 4) that does not have CuCl2 to six samples containing 94 &#956;L of human serum to use as controls.</li>
		<li>Incubate human serum samples for 24 h in an oven at 37 &#176;C and determine levels of oxidation with TBARS assay (section 4).</li>
		<li>Repeat steps 1.3.2&#8211;1.3.4 2x on two separate days to determine reproducibility of the TBARS assay.</li>
	</ol>
	</li>
</ol>
2. Reagent preparation
CAUTION: Thiobarbituric acid causes skin and eye irritation and maybe harmful by inhalation or skin absorption. Acetic acid can damage internal organs if inhaled. Prepare all acid solutions in a fume hood.
<ol>
	<li>Preparation of 8.1% (w/v) sodium dodecylsulfate (SDS) solution
	<ol>
		<li>Weight out 32.4 g of SDS and dissolve in 350 mL of DI water in a beaker. Use a magnetic stir bar to gently dissolve SDS and avoid making bubbles. Bring final volume to 400 mL with DI water and store SDS solution at RT. 
		NOTE: Here, excess 8.1% SDS solution is prepared; however, for 96 samples, only about 20 mL of the 8.1% SDS solution are needed. Prepare this solution according to the number of samples being analyzed.</li>
	</ol>
	</li>
	<li>Preparation of 3.5 M sodium acetate buffer (pH = 4)
	<ol>
		<li>Dilute 100 mL of glacial acetic acid in 350 mL of DI water in a beaker. Use a magnetic stir bar to gently dissolve it.</li>
		<li>Prepare a 6.5 M NaOH solution using sodium hydroxide beads in water. Dissolve 13 g of NaOH beads in 40 mL of DI water and bring to a final volume of 50 mL with DI water.</li>
		<li>Slowly add about 46 mL of the 6.5 M NaOH solution to the acetic acid solution while mixing with the stir bar (this should raise the pH to 4, but confirm by slowly adding the NaOH solution while measuring using a pH meter).</li>
		<li>Bring final volume to 500 mL with DI water and store sodium acetate buffer at RT.</li>
	</ol>
	</li>
	<li>Preparation of 0.8% aqueous solution of thiobarbituric acid (adjusted to pH = 4) 
	NOTE: In this step, preparation of thiobarbituric acid is optimized for large volumes, since a large number of samples is going to be analyzed (108 samples, not including the standards). Prepare this solution depending on the number of samples planned for analysis.
	<ol>
		<li>Prepare a 5 M sodium hydroxide solution using sodium hydroxide beads and water: dissolve 4 g of sodium hydroxide beads in a final volume of 20 mL of water. Store in a plastic container. This solution should be freshly prepared for each batch.</li>
		<li>Weight 4 g of thiobarbituric acid and add 450 mL of DI water. Use a magnetic stir bar to gently dissolve it. 
		NOTE: This solution will eventually be brought to a 500 mL total volume.</li>
		<li>While dissolving thiobarbituric acid with a stir bar, add (slowly and in a dropwise manner) about 3 mL of the 5 M NaOH solution in 100 &#956;L increments. After adding the NaOH solution, the thiobarbituric acid particles will start to dissolve.</li>
		<li>If the thiobarbituric acid particles still have not fully dissolved, add more of the 5 M NaOH solution in 100 &#956;L increments until all thiobarbituric acid particles are fully dissolved. For this particular volume of solution, a total of 4 mL of the 5 M NaOH solution is added to fully dissolve the thiobarbituric acid particles. 
		NOTE: At this concentration, thiobarbituric acid will not fully dissolve unless the pH is nearly 4.</li>
		<li>Stop adding NaOH after all the thiobarbituric&#160;acid has fully dissolved. Avoid exceeding a pH of 4. The final pH can be verified by taking 1 &#956;L from the mixing thiobarbituric&#160;acid solution and placing it onto pH paper.</li>
		<li>Bring final volume to 500 mL with DI water and store aqueous 0.8% thiobarbituric acid solution at RT.</li>
	</ol>
	</li>
</ol>
3. Malondialdehyde bis(dimethyl acetal) standard sample preparation
NOTE: Malondialdehyde (MDA) is unstable and not commercially available. However, there are different chemical forms of MDA that are commercially available, such as MDA tetrabutylammonium salt, MDA bis(dimethyl acetal), and MDA bis(diethyl acetal). Of these three chemical forms, MDA bis(dimethyl acetal) is used here, because a majority of studies use this same standard21,22. If choosing to use the other two chemical forms of MDA, prior validation of their suitability should be carried out.
<ol>
	<li>Prepare a 550 &#956;M MDA bis(dimethyl acetal) stock solution by diluting 92 &#956;L of pure MDA bis(dimethyl acetal) in 1 L of DI water. Use a magnetic stir bar to mix the solution thoroughly for 10 min. Store solution at 4 &#176;C and use within 1 month.</li>
	<li>Prepare a 200 &#956;M MDA bis(dimethyl acetal) by diluting 726 &#956;L from the 550 &#956;M MDA bis(dimethyl acetal) stock in 1274 &#956;L of DI water. This 200 &#956;M MDA bis(dimethyl acetal) solution should be prepared fresh every time a TBARS assay is performed.</li>
	<li>Standard curve preparation: take eight 2 mL snap-cap polypropylene tubes and label them with letters A through H. Add MDA bis(dimethyl acetal) from the 200 &#956;M stock and dilute in water as described in Table 1.</li>
	<li>Take eight glass tubes (13 mm x 100 mm) and label them A&#8211;H, then add 100 &#956;L of standard to the corresponding tubes. Perform six replicates for the blank standard (sample A) to calculate the limits of detection of the TBARS assay. 
	NOTE: The protocol can be paused here for no more than 1 h.</li>
</ol>
4. TBARS assay
NOTE: Once the TBARS assay is started, it should be finished without stopping.
<ol>
	<li>Take as many glass tubes as needed for the number of samples to be analyzed and label them with the names of the samples. Then, add 100 &#956;L of each prepared sample (as described above) to each glass tube.</li>
	<li>Add 200 &#956;L of 8.1% SDS to each sample and standard and gently swirl the glass tube in a circular motion to mix the sample.</li>
	<li>Add 1.5 mL of the 3.5 M sodium acetate buffer (pH = 4) to each sample and standard.</li>
	<li>Add 1.5 mL of the aqueous 0.8% thiobarbituric acid solution (pH = 4) to each sample and standard.</li>
	<li>Bring the final volume to 4 mL for each sample and standard by adding 700 &#956;L of DI water.</li>
	<li>Tightly cap each glass tube and incubate in a heating block set to 95 &#176;C for 1 h. Cover the glass tubes with aluminum foil to prevent condensation at the tops of the tubes.</li>
	<li>Remove the glass tubes from the heating block and incubate on ice for 30 min.</li>
	<li>Centrifuge samples and standards at 1500 x g for 10 min at 4 &#176;C. After centrifugation, keep the glass tubes containing the samples and standards at RT. 
	NOTE: Keeping the samples on ice or at 4 &#176;C will cause the entire sample or standard to precipitate.</li>
	<li>Immediately after centrifugation, aliquot 150 &#956;L of supernatant from each tube and place into a separate well of a 96 well plate.</li>
	<li>Remove any bubbles from each well using a pipette tip. 
	NOTE: The presence of bubbles will yield inconsistent absorbance readings, leading to high assay imprecision.</li>
	<li>Read absorbances at 532 nm. Subtract the average absorbance reading of the blank samples from all other absorbance readings.</li>
	<li>Create a standard curve by plotting the blank-subtracted absorbance readings at 532 nm vs. the known concentration of each standard. Fit the data points using linear regression. Calculate unknown sample concentrations by using the equation of the linear regression line obtained from the standard curve.</li>
</ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sampling,+standardization,+and+calibration.">Sampling, standardization, and calibration.</a> Fundamentals of Analytical Chemistry. 9th ed. , 153-196 (2014).</li><li>Skoog, D. A., Holler, F. J., Crouch, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction.">Introduction.</a> Principles of Instrumental Analysis. 6th ed. , 1-24 (2007).</li><li>Seibig, S., Van Eldik, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+[FeII(edta)]+Oxidation+by+Molecular+Oxygen+Revisited.+New+Evidence+for+a+Multistep+Mechanism.">Kinetics of [FeII(edta)] Oxidation by Molecular Oxygen Revisited. New Evidence for a Multistep Mechanism.</a> Inorganic Chemistry. 36 (18), 4115-4120 (1997).</li><li>Jeffs, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delta-S-Cys-Albumin:+A+Lab+Test+that+Quantifies+Cumulative+Exposure+of+Archived+Human+Blood+Plasma+and+Serum+Samples+to+Thawed+Conditions.">Delta-S-Cys-Albumin: A Lab Test that Quantifies Cumulative Exposure of Archived Human Blood Plasma and Serum Samples to Thawed Conditions.</a> Molecular &amp; Cellular Proteomics. 18 (10), 2121-2137 (2019).</li><li>Yagi, K., Armstrong, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+Assay+for+the+Level+of+Total+Lipid+Peroxides+in+Serum+or+Plasma.">Simple Assay for the Level of Total Lipid Peroxides in Serum or Plasma.</a> Free Radical and Antioxidant Protocols. Methods in Molecular Biology. , 101-106 (1998).</li><li>Bernheim, F., Bernheim, M. L. C., Wilbur, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+reaction+between+thiobarbituric+acid+and+the+oxidation+products+of+certain+lipides.">The reaction between thiobarbituric acid and the oxidation products of certain lipides.</a> Journal of Biological Chemistry. 174 (1), 257-264 (1948).</li><li>Wilbur, K. M., Bernheim, F., Shapiro, O. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+thiobarbituric+acid+reagent+as+a+test+for+the+oxidation+of+unsaturated+fatty+acids+by+various+agents.">The thiobarbituric acid reagent as a test for the oxidation of unsaturated fatty acids by various agents.</a> Archives of Biochemistry. 24 (2), 305-313 (1949).</li><li>Kwon, T. W., Watts, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+malonaldehyde+by+ultraviolet+spectrophotometry.">Determination of malonaldehyde by ultraviolet spectrophotometry.</a> Journal of Food Science. 28 (6), 627-630 (1963).</li><li>Esterbauer, H., Schaur, F. J., Zollner, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemistry+and+biochemistry+of+4-hydroxynonenal,+malonaldehyde+and+related+aldehydes.">Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes.</a> Free Radical Biology &amp; Medicine. 11 (1), 81-128 (1991).</li><li>Dalle-Donne, I., Rossi, R., Colombo, R., Giustarini, D., Milzani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomarkers+of+oxidative+damage+in+human+disease.">Biomarkers of oxidative damage in human disease.</a> Clinical Chemistry. 52 (4), 601-623 (2006).</li><li>Jentzsch, A. M., Bachmann, H., F&#252;rst, P., Biesalski, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+analysis+of+malondialdehyde+in+human+body+fluids.">Improved analysis of malondialdehyde in human body fluids.</a> Free Radical Biology and Medicine. 20 (2), 251-256 (1996).</li><li>Jo, C., Ahn, D. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorometric+Analysis+of+2-Thiobarbituric+Acid+Reactive+Substances+in+Turkey.">Fluorometric Analysis of 2-Thiobarbituric Acid Reactive Substances in Turkey.</a> Poultry Science. 77 (3), 475-480 (1998).</li><li>Tsikas, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development,+validation+and+biomedical+applications+of+stable-isotope+dilution+GC-MS+and+GC-MS/MS+techniques+for+circulating+malondialdehyde+(MDA)+after+pentafluorobenzyl+bromide+derivatization:+MDA+as+a+biomarker+of+oxidative+stress+and+its+relation+to+15(S)-8-iso-prostaglandin+F2&#945;+and+nitric+oxide+(NO).">Development, validation and biomedical applications of stable-isotope dilution GC-MS and GC-MS/MS techniques for circulating malondialdehyde (MDA) after pentafluorobenzyl bromide derivatization: MDA as a biomarker of oxidative stress and its relation to 15(S)-8-iso-prostaglandin F2&#945; and nitric oxide (NO).</a> Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences. 1019, 95-111 (2016).</li><li>Barden, A. E., Mas, E., Croft, K. D., Phillips, M., Mori, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimizing+artifactual+elevation+of+lipid+peroxidation+products+(F+2-isoprostanes)+in+plasma+during+collection+and+storage.">Minimizing artifactual elevation of lipid peroxidation products (F 2-isoprostanes) in plasma during collection and storage.</a> Analytical Biochemistry. 449 (1), 129-131 (2014).</li><li>Jeffs, J. W., Ferdosi, S., Yassine, H. N., Borges, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+instability+of+glycated+albumin:+A+role+for+autoxidative+glycation.">Ex vivo instability of glycated albumin: A role for autoxidative glycation.</a> Archives of Biochemistry and Biophysics. 629, 36-42 (2017).</li><li>Lee, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malondialdehyde+in+Stored+Plasma.">Malondialdehyde in Stored Plasma.</a> Biochemical and Biophysical Research Communications. 95 (4), 1663-1672 (1980).</li><li>Tsikas, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+GC-MS/MS+measurement+of+malondialdehyde+and+4-hydroxy-2-nonenal+in+human+plasma:+Effects+of+long-term+L-arginine+administration.">Simultaneous GC-MS/MS measurement of malondialdehyde and 4-hydroxy-2-nonenal in human plasma: Effects of long-term L-arginine administration.</a> Analytical Biochemistry. 524, 31-44 (2017).</li></ol>

The authors have no competing financial interests or other conflicts of interest to disclose.

Despite its limitations1,3,4,7,8,9,10,12,13,14,15,19 and a lack of suitability for comparison between laboratories, t...

Lipid peroxidation is a process in which free radicals, such as reactive oxygen species and reactive nitrogen species, attack carbon-carbon double bonds in lipids, a process that involves the abstraction of a hydrogen from a carbon and insertion of an oxygen molecule. This process leads to a mixture of complex products including, lipid peroxyl radicals, and hydroperoxides as the primary products, as well as malondialdehyde (MDA) and 4-hydroxynonenal as predominant secondary products1.
MDA has been widely used in biomedical research as a marker of lipid peroxidation due to its facile reaction with thiobarbituric acid (TBA). The reaction leads to the formation of MDA-TBA2, a conjugate that absorbs in the visible spectrum at 532 nm and produces a red-pink color2. Other molecules derived from lipid peroxidation besides MDA can also react with TBA and absorb light at 532 nm, contributing to the overall absorption signal that is measured. Similarly, MDA can react with most other major classes of biomolecules, potentially limiting its accessibility for reaction with TBA3,4. As such, this traditional assay is simply considered to measure &#8220;thiobarbituric acid reactive substances&#8221; or TBARS5.
When correctly applied and interpreted, the TBARS assay is generally considered a good indicator of the overall levels of oxidative stress in a biological sample6. Unfortunately, as documented by Khoubnasabjafari and others, the TBARS assay is often conducted and interpreted in ways that facilitate dubious conclusions3,4,7,8,9,10,11. The causes for this are rooted primarily in sample-related pre-analytical variables and a lack of assay ruggedness that prohibits seemingly minor variations in assay protocol without substantial changes in assay results1,7,12,13.
Preanalytical variables related to biospecimen handling and storage (e.g., blood plasma kept temporarily at -20 &#176;C)14,15 can have a major impact on TBARS assay results16,17; so much so, that TBARS assay results should not be compared across different laboratories unless warranted by explicit interlaboratory analytical validation data. This recommendation is akin to how western blots are commonly used and interpreted. Comparisons of band densities are valid for within-blot and perhaps within-laboratory studies, but comparing band densities between laboratories is generally considered an invalid practice.
Some researchers have suggested that MDA as measured by the TBARS assay simply does not meet the analytical or clinical criteria required of an acceptable biomarker3,9,10,18,19. Indeed, if the assay had not been developed over 50 years ago, it probably would not have gained the widespread use and tacit acceptability that it has today. Although there are other assays with greater analytical sensitivity, specificity, and ruggedness used for determining oxidative stress, TBARS assay based on absorbance at 532 nm remains by far one of the most commonly used assays for the determination of lipid peroxidation20, and thereby assessment of oxidative stress.
The TBARS assay can only be found as an expensive kit (over 400 U.S. dollars), in which the instructions do not provide detailed information on most concentrations of the reagents used. Additionally, the reagents provided can only be used for one experiment, because only one colorimetric standard curve can be made per kit. This can be problematic for researchers who intend to determine levels of oxidation within a few samples at different timepoints, because the same standard curve cannot be used at multiple times. Hence, multiple kits need to be purchased for multiple experiments. Currently, unless an expensive kit is purchased, there is not a detailed protocol available for how to perform a TBARS assay. Some researchers in the past have vaguely described how to perform a TBARS assay21,22, but neither a fully detailed protocol or comprehensive video on how to conduct the TBARS assay without an expensive kit is available in the literature.
Here we report a detailed, analytically validated for-purpose methodology on how to perform a TBARS assay in a simple, reproducible, and inexpensive way. Changes in the lipid peroxidation of human serum, HepG2 lysates, and low density lipoproteins upon treatment with Cu(II) ions are demonstrated as illustrative applications for the TBARS assay. Results demonstrate that this TBARS assay is consistent and reproducible on a day-to-day basis.

<table><tbody><tr><td>1x Sterile PBS pH 7.4 1 L</td><td>VWR, PA</td><td>101642--262</td><td>cell lysis reagent</td></tr><tr><td>50 mL self-standing centrifuge tube</td><td>Corning, NY</td><td>CLS430897</td><td>General material</td></tr><tr><td>96 well plate, Non-Treated, clear, with lid, Non-sterile</td><td>Thermo Fisher Scientific, MA</td><td>280895</td><td>To measure absorbance</td></tr><tr><td>Amicon Ultra-0.5 100 kD centrifugal spin filter device</td><td>Fisher Scientific, NH</td><td>UFC510024</td><td>LDL purification</td></tr><tr><td>Caps for glass tubes</td><td>Thermo Fisher Scientific, MA</td><td>14-930-15D</td><td>for TBARS assay</td></tr><tr><td>Copper II Chloride</td><td>SIGMA, MO</td><td>222011-250G</td><td>to induce oxidation</td></tr><tr><td>Culture tubes, Disposable, with Screw-Cap Finish, Borosilicate Glass (13 x 100 mm)</td><td>VWR, PA</td><td>53283-800</td><td>for TBARS assay</td></tr><tr><td>Eagle&#039;s Minimum Essential Medium (EMEM)</td><td>ATCC, VA</td><td>HB-8065</td><td>HepG2 cell media</td></tr><tr><td>Eppendorf Safe-Lock Tubes, 1.5 mL</td><td>eppendorf, NY</td><td>22363204</td><td>General material</td></tr><tr><td>Eppendorf Safe-Lock Tubes, 2.0 mL</td><td>Genesee Sceitific, CA</td><td>22363352</td><td>General material</td></tr><tr><td>Fetal Bovine Serum US Source</td><td>Omega Scientific, CA</td><td>FB-11</td><td>for cell culture</td></tr><tr><td>Glacial Acetic Acid</td><td>SIGMA, MO</td><td>27225-1L-R</td><td>TBARS Reagent</td></tr><tr><td>Halt Protease Inhibitor Cocktail (100x)</td><td>Thermo Scientific, MA</td><td>87786</td><td>cell lysis reagent</td></tr><tr><td>HEPES</td><td>SIGMA, MO</td><td>H3375-250G</td><td>LDL solvent</td></tr><tr><td>HepG2 Cells</td><td>ATCC, VA</td><td>HB-8065</td><td>Biological matrix prototype</td></tr><tr><td>Hydrocloric acid (HCl)</td><td>Fisher Scientific, NH</td><td>A144-212</td><td>cell lysis reagent</td></tr><tr><td>Legend Micro 17 Centrifuge</td><td>Thermo Scientific, MA</td><td>75002431</td><td>General material</td></tr><tr><td>Low Density Lipoprotein, Human Plasma</td><td>Athens Research &amp;amp; Technology, GA</td><td>12-16-120412</td><td>Biological matrix prototype</td></tr><tr><td>Magnetic Stir Bars, Octagon 6-Assortment</td><td>VWR, PA</td><td>58948-025</td><td>General material</td></tr><tr><td>Malondialdehyde bis (dimethyl acetal)</td><td>SIGMA, MO</td><td>8207560250</td><td>TBARS Standard</td></tr><tr><td>Multiskan Go Microplate Spectrophotometer</td><td>Fisher Scientific, NH</td><td>51119200</td><td>To measure absorbance</td></tr><tr><td>NP-40</td><td>EMD Millipore Corp, MA</td><td>492016-100ML</td><td>cell lysis reagent</td></tr><tr><td>Sodium Chloride</td><td>SIGMA, MO</td><td>S7653-1KG</td><td>cell lysis reagent</td></tr><tr><td>Sodium dodecyl sulfate (SDS)</td><td>SIGMA, MO</td><td>436143-100G</td><td>TBARS Reagent</td></tr><tr><td>Sodium hydroxide</td><td>SIGMA, MO</td><td>367176-2.5KG</td><td>TBARS Reagent</td></tr><tr><td>SpeedVac Concentrator</td><td>Thermo Scientific, MA</td><td>SC250EXP</td><td>For concentrating cell lysates</td></tr><tr><td>T-75 Flask, Tissue Culture Treated, 250 mL, w/filter cap</td><td>USA Scientific, FL</td><td>658175</td><td>cell culture</td></tr><tr><td>Thiobarbituric Acid</td><td>SIGMA, MO</td><td>T5500-100G</td><td>TBARS Reagent</td></tr><tr><td>TRIS base</td><td>Fluka, GA</td><td>93362</td><td>cell lysis reagent</td></tr><tr><td>Trypsin (1x)</td><td>VWR, PA</td><td>16777-166</td><td>To detach HepG2 cells</td></tr></tbody></table>

evaluation of oxidative stress in biological samples using the thiobarbituric acid reactive substances assay

Despite its limited analytical specificity and ruggedness, the thiobarbituric acid reactive substances (TBARS) assay has been widely used as a generic metric of lipid peroxidation in biological fluids. It is often considered a good indicator of the levels of oxidative stress within a biological sample, provided that the sample has been properly handled and stored. The assay involves the reaction of lipid peroxidation products, primarily malondialdehyde (MDA), with thiobarbituric acid (TBA), which leads to the formation of MDA-TBA2 adducts called TBARS. TBARS yields a red-pink color that can be measured spectrophotometrically at 532 nm. The TBARS assay is performed under acidic conditions (pH = 4) and at 95 &#176;C. Pure MDA is unstable, but these conditions allow the release of MDA from MDA bis(dimethyl acetal), which is used as the analytical standard in this method. The TBARS assay is a straightforward method that can be completed in about 2 h. Preparation of assay reagents are described in detail here. Budget-conscious researchers can use these reagents for multiple experiments at a low cost rather than buying an expensive TBARS assay kit that only permits construction of a single standard curve (and thus can only be used for one experiment). The applicability of this TBARS assay is shown in human serum, low density lipoproteins, and cell lysates. The assay is consistent and reproducible, and limits of detection of 1.1 &#956;M can be reached. Recommendations for the use and interpretation of the spectrophotometric TBARS assay are provided.

Under acidic conditions (pH = 4) and at 95 &#176;C, malondialdehyde (MDA) bis(dimethyl acetal) yields MDA23. MDA and closely related chemical congeners react with two molecules of thiobarbituric acid (TBA) to produce compounds called thiobarbituric acid reactive substances (TBARS), which give a red-pink color and have an absorbance &#955;max at 532 nm (Figure 1, Figure 2). Using MDA bis (dimethyl acetal) as the standard, standa...

Watch this Scientific Journal Video about Evaluation of Oxidative Stress in Biological Samples Using the Thiobarbituric Acid Reactive Substances Assay at JoVE.com

Evaluation of Oxidative Stress in Biological Samples Using the Thiobarbituric Acid Reactive Substances Assay

The goal of the thiobarbituric acid reactive substances assay is to assess oxidative stress in biological samples by measuring the production of lipid peroxidation products, primarily malondialdehyde, using visible wavelength spectrophotometry at 532 nm. The method described here can be applied to human serum, cell lysates, and low density lipoproteins.

Despite its limited analytical specificity and ruggedness, the thiobarbituric acid reactive substances (TBARS) assay has been widely used as a generic ...

evaluation-oxidative-stress-biological-samples-using-thiobarbituric

Universidad San Sebastian

Research

JoVE Journal

Chemistry

28.0K Views.  Arizona State University. The goal of the thiobarbituric acid reactive substances assay is to assess oxidative stress in biological samples by measuring the production of lipid peroxidation products, primarily malondialdehyde, using visible wavelength spectrophotometry at 532 nm. The method described here can be applied to human serum, cell lysates, and low density lipoproteins.

Video: Evaluation of Oxidative Stress in Biological Samples Using the Thiobarbituric Acid Reactive Substances Assay

Imaging Approaches to Assessments of Toxicological Oxidative Stress Using Genetically-encoded Fluorogenic Sensors

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including destruction of the sample, introduction of potential artifacts, and a lack of specificity for the reactive species involved. Thus, there is a current need in the field of toxicology for non-destructive, sensitive, and specific methods that can be used to observe and quantify intracellular redox perturbations, more commonly referred to as oxidative stress. Here, we present a method for the use of two genetically-encoded fluorogenic sensors, roGFP2 and HyPer, to be used in live-cell imaging studies to observe xenobiotic-induced oxidative responses. roGFP2 equilibrates with the glutathione redox potential (EGSH), while HyPer directly detects hydrogen peroxide (H2O2). Both sensors can be expressed into various cell types via transfection or transduction, and can be targeted to specific cellular compartments. Most importantly, live-cell microscopy using these sensors offers high spatial and temporal resolution that is not possible using conventional methods. Changes in the fluorescence intensity monitored at 510 nm serves as the readout for both genetically-encoded fluorogenic sensors when sequentially excited by 404 nm and 488 nm light. This property makes both sensors ratiometric, eliminating common microscopy artifacts and correcting for differences in sensor expression between cells. This methodology can be applied across a variety of fluorometric platforms capable of exciting and collecting emissions at the prescribed wavelengths, making it suitable for use with confocal imaging systems, conventional wide-field microscopy, and plate readers. Both genetically-encoded fluorogenic sensors have been used in a variety of cell types and toxicological studies to monitor cellular EGSH and H2O2 generation in real-time. Outlined here is a standardized method that is widely adaptable across cell types and fluorometric platforms for the application of roGFP2 and HyPer in live-cell toxicological assessments of oxidative stress.

This manuscript describes the use of genetically-encoded fluorogenic reporters in an application of live-cell imaging for the examination of xenobiotic-induced oxidative stress. This experimental approach offers unparalleled spatiotemporal resolution, sensitivity, and specificity while avoiding many of the shortcomings of conventional methods used for the detection of toxicological oxidative stress.

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including ...

Cancer Research

Detection of Total Reactive Oxygen Species in Adherent Cells by 2',7'-Dichlorodihydrofluorescein Diacetate Staining

Oxidative stress is an important event under both physiological and pathological conditions. In this study, we demonstrate how to quantify oxidative stress by measuring total reactive oxygen species (ROS) using 2&#39;,7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA) staining in colorectal cancer cell lines as an example. This protocol describes detailed steps including preparation of DCFH-DA solution, incubation of cells with DCFH-DA solution, and measurement of normalized intensity. DCFH-DA staining is a simple and cost-effective way to detect ROS in cells. It can be used to measure ROS generation after chemical treatment or genetic modifications. Therefore, it is useful for determining cellular oxidative stress upon environment stress, providing clues to mechanistic studies.

Here, we present a protocol to detect total cellular reactive oxygen species (ROS) using 2&#39;,7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA). This method can visualize cellular ROS localization in adherent cells with a fluorescence microscope and quantify ROS intensity with a fluorescence plate reader. This protocol is simple, efficient and cost-effective.

Oxidative stress is an important event under both physiological and pathological conditions. In this study, we demonstrate how to quantify oxidative ...

Biochemistry

Ecotoxicological Methodologies to Evaluate Biomarkers at Different Scales in Neotropical Anurans

The new questions in ecotoxicology highlight the importance of applying a battery of biomarkers, as this results in ecotoxicological predictions that improve not only the interpretation of the effects of environmental stressors on organisms but also the determination of their possible impact. It is well known that the use of ecotoxicological biomarkers at different levels of organization allows for the prediction of the biological responses of organisms to environmental stressors, which is useful in environmental risk assessment. 
Nevertheless, it is necessary to consider the optimization of basic procedures, to generate historical data in control groups, and to employ specific bioassays to evaluate responses in organs and tissues in order to elucidate the nature and variation of the effects observed. Therefore, the present work aims to describe several ecotoxicological methodologies employed in all stages of neotropical anurans at different ecological levels and to validate them as useful biomarkers to be used both in wildlife and in laboratory conditions. In this work, these biomarkers were applied at the individual/organismic level (body condition index), histological/physiological level (histopathology, histometric, and pigmentary analyses), biochemical level (oxidative stress enzymes), and genetic level (direct and oxidative damage in DNA by comet assay). 
Although these methodologies have small variations or modifications depending on the species, these techniques provide effective biomarkers for evaluating the effect of xenobiotics on anurans, which possess certain characteristics that make them useful indicator species of aquatic and terrestrial ecosystems. In conclusion, the battery of biomarkers employed in the present study has proven to be adequate for estimating toxic responses in Neotropical anurans and can be further recommended as bioindicators for identifying the impact of pollutants on the aquatic ecosystems of the region. Finally, it is recommended to achieve the standardization of these important biomarkers for anurans in specific regions as well as to possibly include them in risk assessments and decision-making.

In this paper, standardized ecotoxicological methods for the evaluation of biomarkers in neotropical anuran species are presented. Specifically, this paper details several methodologies at different scales of ecotoxicological evaluation, such as the genetic, cellular-histological, biochemical, morphological, and individual levels.

The new questions in ecotoxicology highlight the importance of applying a battery of biomarkers, as this results in ecotoxicological predictions that ...

Environment

Hellgrammite as a Non-Conventional Biomonitor for Surface Water and Environmental Health Assessment

The larvae of dobsonflies from the genus Corydalus, commonly known as Hellgrammites, are characterized by their notable size, extensive range of occurrence, and extended period of immaturity, which can last up to one year. Hellgrammites are clearly known to exhibit sensitivity to pollution and habitat structure impacts. Given these unique features, using Corydalus texanus larvae is highly suitable as reliable biomonitoring agents to assess the ecological integrity of aquatic ecosystems. This protocol aims to provide the necessary tools for C. texanus assessment and demonstrate their efficacy through a case study. Research findings have practical implications, indicating that C. texanus larvae exhibit early-warning responses to mining pollution, bioaccumulating high amounts of heavy metals such as Zn, Fe, and Al. The presence or absence of C. texanus populations may serve as a helpful indicator for identifying potential issues related to ecosystem health. The unconventional approach has shown early warnings of pollution in mining-impacted sites, highlighting the need for timely action to protect the environment. Given their unique traits, the use of C. texanus larvae is highly suggested as a reliable non-conventional bioindicator.

This protocol describes the use of biomarkers for the early detection of deleterious impacts in aquatic ecosystems. The biomarkers are closely related to sentinel traits, and their changes aid in detecting early-warning damages.

The larvae of dobsonflies from the genus Corydalus, commonly known as Hellgrammites, are characterized by their notable size, extensive range of ...

Resin-Assisted Capture Coupled with Isobaric Tandem Mass Tag Labeling for Multiplexed Quantification of Protein Thiol Oxidation

Reversible oxidative modifications on protein thiols have recently emerged as important mediators of cellular function. Herein we describe the detailed procedure of a quantitative redox proteomics method that utilizes resin-assisted capture (RAC) in combination with tandem mass tag (TMT) isobaric labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to allow multiplexed stochiometric quantification of oxidized protein thiols at the proteome level. The site-specific quantitative information on oxidized cysteine residues provides additional insight into the functional impacts of such modifications. 
The workflow is adaptable across many sample types, including cultured cells (e.g., mammalian, prokaryotic) and whole tissues (e.g., heart, lung, muscle), which are initially lysed/homogenized and with free thiols being alkylated to prevent artificial oxidation. The oxidized protein thiols are then reduced and captured by a thiol-affinity resin, which streamlines and simplifies the workflow steps by allowing the proceeding digestion, labeling, and washing procedures to be performed without additional transfer of proteins/peptides. Finally, the labeled peptides are eluted and analyzed by LC-MS/MS to reveal comprehensive stoichiometric changes related to thiol oxidation across the entire proteome. This method greatly improves the understanding of the role of redox-dependent regulation under physiological and pathophysiological states related to protein thiol oxidation.

Protein thiol oxidation has significant implications under normal physiological and pathophysiological conditions. We describe the details of a quantitative redox proteomics method, which utilizes resin-assisted capture, isobaric labeling, and mass spectrometry, enabling site-specific identification and quantification of reversibly oxidized cysteine residues of proteins.

Reversible oxidative modifications on protein thiols have recently emerged as important mediators of cellular function. Herein we describe the detailed ...

GSH/GSSG Measurement &#8211; A Refined OPA Assay for Antioxidant Research

Rapid Quantification of Oxidized and Reduced Forms of Glutathione Using Ortho -phthalaldehyde in Cultured Mammalian Cells In Vitro

Glutathione has long been considered a key biomarker for determining the antioxidant response of the cell. Hence, it is a primary marker for reactive oxygen species studies. The method utilizes Ortho-phthalaldehyde (OPA) to quantify the cellular concentration of glutathione(s). OPA conjugates with reduced glutathione (GSH) via sulfhydryl binding to subsequently form an isoindole, resulting in a highly fluorescent conjugate. To attain an accurate result of both oxidized glutathione (GSSG) and GSH, a combination of masking agents and reducing agents, which have been implemented in this protocol, are required. Treatments may also impact cellular viability. Hence, normalization via protein assay is presented in this multiparametric assay. The assay demonstrates a pseudo-linear detection range of 0.234 - 30&#181;M (R2=0.9932&#177;0.007 (N=12)) specific to GSH. The proposed assay also allows for the determination of oxidized glutathione with the addition of the masking agent N-ethylmaleimide to bind reduced glutathione, and the reducing agent tris(2-carboxyethyl) phosphine is introduced to cleave the disulfide bond in GSSG to produce two molecules of GSH. The assay is used in combination with a validated bicinchoninic acid assay for protein quantification and an adenylate kinase assay for cytotoxicity assessment.

Author Spotlight: Innovating Thiol Quantification and Biomarker Detection for Oxidative Stress Research 

Quantification of both oxidized and reduced forms of glutathione (GSSG and GSH, respectively) has been achieved through the use of Ortho-phthalaldehyde (OPA). OPA becomes highly fluorescent once conjugated to GSH but is unable to conjugate GSSG until reduced. Here, we describe a multiparametric assay to quantify both using protein quantification for normalization.

Glutathione has long been considered a key biomarker for determining the antioxidant response of the cell. Hence, it is a primary marker for reactive ...

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant1,2. The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato3. Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas3. Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)4. DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue4. ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins5. Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes6,7. Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry8,9. The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter10. After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.

Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on ...

Biology

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

Thiobarbituric Acid-Reactive Substances Assay to Assess Oxidative Stress in Human Serum

Source: Borges, C. R. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/61122">Evaluation of Oxidative Stress in Biological Samples Using the Thiobarbituric Acid Reactive Substances Assay.</a> J. Vis. Exp. (2020)
This video demonstrates the thiobarbituric acid reactive substances (TBARS) assay to assess oxidative stress in biological samples.

Source: Borges, C. R. et al., Evaluation of Oxidative Stress in Biological Samples Using the Thiobarbituric Acid Reactive Substances Assay. J. Vis. Exp. ...

JoVE Encyclopedia of Experiments

Biological Techniques

Assay Techniques

Biochemical assays

A Spot Plate Assay for Evaluating Oxidative Stress Responses in Bacterial Strains

To begin this procedure, set up overnight cultures of the strains to be tested in LB, with and without NTBC. On the following day, prepare LB agar plates containing hydrogen peroxide as an oxidative stressor. First, melt the LB agar flasks and cool the media to approximately 50 degrees Celsius at room temperature.
Next, add hydrogen peroxide directly to the cooled media at the desired concentrations. Swirl the flask to mix. A range of hydrogen peroxide concentrations from 0 to 1 millimolar is a good starting point. Pour the plates immediately, and flame the surface to remove bubbles. The yield is four plates per 100 millimeters of LB agar. Mark the plates with the hydrogen peroxide concentration.
Place the plates uncovered in a biological flow hood, with a fan running for 30 minutes to remove excess moisture from the plates. The plates should be used on the same day they are prepared. While the plates are drying, wash and measure the OD600 of the overnight cultures.
Normalize the OD600 of all the overnight cultures to the lowest value for the set of strains being tested. To do this, determine the volume of culture needed to dilute the culture to the lowest OD600 in a total volume of 1 milliliter.
For example, if a culture has an OD600 of 3 and the lowest OD600 for the set of strains is 2.5, perform the following calculation, which will result in 0.833 milliliters as the volume of culture needed. Place 0.833 milliliters of culture in a microfuge tube. Next, calculate the amount of LB plus NTBC or LB plus DMSO needed to bring the culture volume to 1 milliliter. In this example, the amount would be 0.167 milliliters. Mix by vortexing.
To maintain cultures in a constant concentration of NTBC or DMSO, perform 10-fold serial dilutions of the normalized overnight cultures in PBS plus NTBC or PBS plus DMSO. Make stock solutions of PBS plus NTBC and PBS plus DMSO scaling up or down, depending on how many strains are tested.
For one set of dilutions for one strain, mix 300 micromolar of NTBC or an equivalent volume of DMSO with PBS to yield a total volume of 720 microliters. Add 90 microliters of PBS plus NTBC or PBS plus DMSO to the appropriate microfuge tubes labeled for 10 to the minus 1 through 10 to the minus 7 serial dilutions. Add 10 microliters of culture to the appropriate 10 to the minus 1 dilution tube.
Mix by vortexing, and transfer 10 microliters of the 10 to the minus 1 dilution to the 10 to the minus 2 dilution tube. Changing pipette tips between dilutions, repeat until all dilutions have been performed.
Spot 5 microliters of the 10 to the minus 3 through 10 to the minus 7 dilutions on LB plus hydrogen peroxide plates in duplicate for each strain. Use one pipette tip if spots are plated for most dilute to least dilute. Do not tip or tilt the plate, until the liquid has dried into the plate. Incubate the plates at 37 degrees Celsius. For 24 to 48 hours, depending on the strain. Once good-sized colonies have appeared, photograph the plates using a CCD camera above a transilluminator.

Evaluation of Oxidative Stress in Biological Samples Using the Thiobarbituric Acid Reactive Substances Assay

Summary

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Rapid Quantification of Oxidized and Reduced Forms of Glutathione Using Ortho -phthalaldehyde in Cultured Mammalian Cells In Vitro

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Thiobarbituric Acid-Reactive Substances Assay to Assess Oxidative Stress in Human Serum

A Spot Plate Assay for Evaluating Oxidative Stress Responses in Bacterial Strains