Critical Methods

With a vision for establishing standards for methods used redox biology research, SfRBM has published a series of papers highlighting Critical Methods in our journals. Each article provides current standards for methods used in the field with caveats explaining limitations of the methods and reasonable interpretation of the results. Compendium of these articles are provided below and will be updated as new publications become available.


[18F]Fluoro-DCP, a first generation PET radiotracer for monitoring protein sulfenylation in vivo
Saia K, Chen X, Li Z, Zhu C, Shuklab K, Forshaw T, Wu H, Vance S, Pathirannahel B, Madonna M, Dewhirst M, Tsang A, Poole L, Ramanujamd N, King B, Furdui C

Synthesis and cellular evaluation of click-chemistry probes to study the biological effects of alpha, beta-unsaturated carbonyls
Morozzi C, Sauerland M, Gamon L, Manandhar A, Ulven T, Davies M


Quantitative measures for redox signaling
Pillay C, Eagling B, Driscoll S, Rohwer J

Detailed protocol to assess in vivo and ex vivo myeloperoxidase activity in mouse models of vascular inflammation and disease using hydroethidine
Talib J, Maghzal G, Cheng D, Stocker R

Real-time and high-throughput analysis of mitochondrial metabolic states in living cells using genetically encoded NAD+/NADH sensors
Zhao Y, Yang Y

Investigating mitochondrial redox state using NADH and NADPH autofluorescence
Blacker T, Duchen M


Mass spectrometry in studies of protein thiol chemistry and signaling: Opportunities and caveats
Devarie Baez N, Reisz J, Furdui C

Even free radicals should follow some rules: A Guide to free radical research terminology and methodology
Forman HJ, et al.


On the use of fluorescence lifetime imaging and dihydroethidium to detect superoxide in intact animals and ex vivo tissues: a reassessment
Michalski R, Michalowski B, Sikora A, Zielonka J, Kalyanaraman B


Methods of lipid oxidation product identification and quantification
Forman HJ

Quantification of fatty acid oxidation products using online high-performance liquid chromatography tandem mass spectrometry
Levison BS, Zhang R, Wang Z, Fu X, DiDonato JA, Hazen SL

Nitrated fatty acids: synthesis and measurement
Woodcock SR, Bonacci G, Gelhaus SL, Schopfer FJ

Method for the simultaneous determination of free/protein malondialdehyde and lipid/protein hydroperoxides
Grintzalis K, Zisimopoulos D, Grune T, Weber D, Georgiou CD

Measurement of F2-isoprostanes and isofurans using gas chromatography-mass spectrometry
Milne GL, Gao B, Terry ES, Zackert WE, Sanchez SC

Lipidomics of oxidized polyunsaturated fatty acids
Massey KA, Nicolaou A

Utilization of fluorescent probes for the quantification and identification of subcellular proteomes and biological processes regulated by lipid peroxidation products
Cummins TD, et al.

Analytical strategies for characterization of oxysterol lipidomes: liver X receptor ligands in plasma
Griffiths WJ, et al.

An overview of the chemistry and biology of reactive aldehydes
Fritz KS, Petersen DR

Strategies for the analysis of chlorinated lipids in biological systems
Wacker BK, Albert CJ, Ford BA, Ford DA

Determination of phospholipase activity of PAF acetylhydrolase
Stafforini DM, McIntyre TM

Detection and isolation of human serum autoantibodies that recognize oxidatively modified autoantigens
Eggleton P, Nissim A, Ryan BJ, Whiteman M, & Winyard PG


Wanted and wanting: Antibody against methionine sulfoxide
Wehr NB & Levine RL

Analytical measurement of discrete hydrogen sulfide pools in biological specimens
Shen X, Peter EA, Bir S, Wang R, & Kevil CG

A simple fluorescence labeling method for studies of protein oxidation, protein modification, and proteolysis.
Pickering AM & Davies KJA

8-Oxo-2′-deoxyguanosine as a biomarker of tobacco-smoking-induced oxidative stress
Mesaros C, Arora JS, Wholer A, Vachani A, & Blair IA

Measuring reactive oxygen and nitrogen species with fluorescent probes: Challenges and limitations
Kalyanaraman B, et al.

A multilevel analytical approach for detection and visualization of intracellular NO production and nitrosation events using diaminofluoresceins
Cortese-Krott MM, et al.


A method for detection of 4-hydroxy-2-nonenal adducts in proteins
Wakita C, Honda K, Shibata T, Akagawa M, & Uchida K

Horseradish peroxidase compound I as a tool to investigate reactive protein-cysteine residues: from quantification to kinetics
Toledo Jr JC, et al.

Identification of the oxidative stress proteome in the brain
Sultana R & Butterfield DA

Identification of oxidized phospholipids by electrospray ionization mass spectrometry and LC–MS using a QQLIT instrument
Spickett CM, Reis A, & Pitt AR

Measurement of plasma hydrogen sulfide in vivo and in vitro
Shen X, et al.

Measuring EGSH and H2O2 with roGFP2-based redox probes
Morgan B, Sobotta MC, & Dick TP


HPLC study of oxidation products of hydroethidine in chemical and biological systems: ramifications in superoxide measurements
Zielonka J, Hardy M, & Kalyanaraman B

Direct chemiluminescence detection of nitric oxide in aqueous solutions using the natural nitric oxide target soluble guanylyl cyclase
Woldman YY, Sun J, Zweier JL, & Khramtsov VV

Quantification of F2-isoprostanes as a reliable index of oxidative stress in vivo using gas chromatography–mass spectrometry (GC-MS) method
Liu W, Morrow JD, & Yin H

Measuring the poise of thiol/disulfide couples in vivo
Jones DP & Liang Y

Making and working with hydrogen sulfide: The chemistry and generation of hydrogen sulfide in vitro and its measurement in vivo: A review
Hughes MN, Centelles MN, & Moore KP

Methods for the determination and quantification of the reactive thiol proteome
Hill BG, Reily C, Oh J-Y, Johnson MS, & Landar A

Methods for imaging and detecting modification of proteins by reactive lipid species
Higdon AN, et al.

Quantification of protein modification by oxidants
Hawkins CL, Morgan PE, & Davies MJ

Immuno-spin trapping of protein and DNA radicals: “Tagging” free radicals to locate and understand the redox process
Gomez-Mejiba SE, et al.

Detection of protein S-nitrosylation with the biotin-switch technique
Forrester MT, Foster MW, Benhar M, & Stamler JS

Critical Methods in Free Radical Biology & Medicine
Forman HJ

Turnover of oxidatively modified proteins: the usage of in vitro and metabolic labeling
Catalgol B & Grune T

Fluorescent detection of α-aminoadipic and γ-glutamic semialdehydes in oxidized proteins
Akagawa M, Suyama K, & Uchida K

Mouse model of liver ischemia and reperfusion injury: method for studying reactive oxygen and nitrogen metabolites in vivo
Abe Y, et al.


Measurement of formamidopyrimidines in DNA
Jaruga P, Kirkali G, & Dizdaroglu M


Fluorescent and luminescent probes for measurement of oxidative and nitrosative species in cells and tissues: Progress, pitfalls, and prospects
Wardman P

Methods to detect nitric oxide and its metabolites in biological samples
Bryan NS & Grisham MB