(ORAC) Activity Assay, Trial Size
Contents
1. Product Manual OxiSelect Oxygen Radical Antioxidant Capacity ORAC Activity Assay Trial Size Catalog Number STA 345 T 48 assays FOR RESEARCH USE ONLY Not for use in diagnostic procedures CELL BIOLABS INC Creating Solutions for Life Science Research Introduction Oxidative stress is a physiological condition where there is an imbalance between concentrations of reactive oxygen species ROS and antioxidants However excessive ROS accumulation will lead to cellular injury such as damage to DNA proteins and lipid membranes The cellular damage caused by ROS has been implicated in the development of many disease states such as cancer diabetes cardiovascular disease atherosclerosis and neurodegenerative diseases Under normal physiological conditions cellular ROS generation is counterbalanced by the action of cellular antioxidant enzymes and other redox molecules Because of their potential harmful effects excessive ROS must be promptly eliminated from the cells by this variety of antioxidant defense mechanisms Antioxidants include both hydrophilic and lipophilic molecules for metabolizing ROS Although the products of ROS induced oxidative stress are extensively used to monitor the effects of oxidative stress it is also important to evaluate the antioxidant capacity of biological fluids cells and extracts The Oxygen Radical Antioxidant Capacity ORAC Assay is a classic tool for measuring the antioxidant capacity of2. 10 1093 gerona gls 159 18 Bailey Downs L C et al 2011 Liver specific knockdown of IGF 1 decreases vascular oxidative stress resistance by impairing the Nrf2 dependent antioxidant response a novel model of vascular aging J Gerontol A Biol Sci Med Sci 10 1093 gerona glr164 19 Ungvari Z et al 2011 Extreme longevity is associated with increased resistance to oxidative stress in Arctica islandica the longest living non colonial animal J Gerontol A Biol Sci Med Sci 10 1093 gerona glr044 20 Ungvari Z et al 2011 Free radical production antioxidant capacity and oxidative stress response signatures in fibroblasts from lewis dwarf rats effects of life span extending peripubertal GH treatment J Gerontol A Biol Sci Med Sci 10 1093 gerona glr004 Warranty These products are warranted to perform as described in their labeling and in Cell Biolabs literature when used in accordance with their instructions THERE ARE NO WARRANTIES THAT EXTEND BEYOND THIS EXPRESSED WARRANTY AND CELL BIOLABS DISCLAIMS ANY IMPLIED WARRANTY OF MERCHANTABILITY OR WARRANTY OF FITNESS FOR PARTICULAR PURPOSE CELL BIOLABS s sole obligation and purchaser s exclusive remedy for breach of this warranty shall be at the option of CELL BIOLABS to repair or replace the products In no event shall CELL BIOLABS be liable for any proximate incidental or consequential damages in connection with the products Contact Information Cell Biolabs Inc 7758 Arj
3. below for reference only This data should not be used to interpret or calculate actual sample results 350 12 300 250 100 uM Trolox oO 200 Blank 5 i 150 g 100 50 0 T T T 13 T T 1 T T T F T 1 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 Time Min Trolox uM Figure 1 ORAC Activity Assay Standard Curve Calculation of Results Note A spreadsheet application or plate reader software can be used to perform the calculations 1 Calculate the area under the curve AUC for each sample and standard using the final assay values and the linear regression formula below The AUC can be calculated from the equation below AUC 1 RFU RFUp RFU2 RFUp RFU3 RFU RFUs50 RFUp RFU 60 RFUp RFUp relative fluorescence value of time point zero RFU relative fluorescence value of time points eg RFUs is relative fluorescence value at minute five Ja CELL BIOLABS INC oN AUC Blank AUC Antioxidant 10 15 20 25 30 35 40 45 50 55 60 0 5 10 15 20 25 30 35 40 45 50 55 60 Time min Time min 2 Calculate the Net AUC by subtracting the Blank AUC from the AUC of each sample and standard Net AUC AUC Antioxidant AUC blank Net AUC 5 10 15 20 25 30 35 40 45 50 55 60 Time min 3 Graph the Net AUC on the y axis against the Trolox Antioxidant Standard concentration on the X axis see Figure 1 4 Calculate the uMole Trolox Equivalents T
4. 0 Table 2 Preparation of Standards for use when testing Lipophilic Samples Note Do not store diluted Antioxidant Standard solutions Assay Protocol Note Each Antioxidant Standard and sample should be assayed in duplicate or triplicate A freshly prepared standard curve should be used each time the assay is performed 1 Add 25 uL of the diluted Antioxidant Standard or samples to the 96 well Microtiter Plate 2 Add 150 uL of the 1X Fluorescein Solution to each well Mix thoroughly Incubate the plate for 30 minutes at 37 C 6 g CELL BIOLABS INC AS 3 4 Add 25 uL of the Free Radical Initiator Solution into each well using either a multichannel pipette or a plate reader liquid handling system Mix the reaction mixture thoroughly by pipetting to ensure homogeneity 5 Immediately begin reading sample and standard wells with a fluorescent microplate reader at 37 C with an excitation wavelength of 480nm and an emission wavelength of 520nm Read the wells in increments between 1 and 5 minutes for a total of 60 minutes Save values for Calculation of Results below Note The final assay values of blank control should be less than 10 of the initial values in order for the assay to be completed Example of Results The following figure demonstrates typical OxiSelect ORAC Activity Assay results One should use the data
5. E of unknown sample by comparing the standard curve Results ORAC value may be expressed as TE per L or g of sample Calculation Example 25 uL of 10 fold diluted sample is assayed along with 25 uL of each Trolox antioxidant standard including blank as described in Assay Protocol The average AUC is 4 3 for blank and 9 1 for sample Net AUC AUC Antioxidant AUC blank 9 1 4 3 4 8 Based on the Trolox antioxidant standard curve the equivalent Trolox concentration is 20 uM therefore ORAC value Sample 20 uM x 10 dilution factor 200 uM TE 200 uMole TE L 8 CELL BIOLABS INC References 1 2 3 Ames B N Shigenaga M K and Hagen T M 1993 Proc Natl Acad Sci USA 90 7915 7922 Cao G and Prior R 1999 Methods Enzymol 299 50 62 Huang D Ou B Hampsch Woodill M Flanagan J and Prior R 2002 J Agric Food Chem 50 4437 4444 Huang D Ou B amp Prior R 2005 J Agric Food Chem 53 1841 1856 5 Ou B Hampsch Woodill M and Prior R 2001 J Agric Food Chem 49 4619 4626 6 Rice Evans C and Miller NJ 1994 Methods Enzymol 234 279 293 Trolox is a trademark of Hoffman LaRoche Recent Product Citations 1 2 3 10 11 12 13 Nishikawa Y et al 2015 Cytoprotective effects of lysophospholipids from sea cucumber Holothuria atra PLoS One 10 e0135701 Gutierrez R M P amp Madrigalez Ahuatzi D 2015
6. Investigating antioxidant properties of the diterpenes from seeds of Phalaris canariensis J Nutr Food Sci doi 10 4172 2155 9600 1000376 Orena S et al 2015 Extracts of fruits and vegetables activate the antioxidant response element in IMR 32 cells J Nutr 145 2006 2011 Gonzalez B et al 2015 Polyphenol anthocyanin and antioxidant content in different parts of maqui fruits Aristotelia chilensis during ripening and conservation treatments after harvest Ind Crop Prod 76 158 165 Okutsu K et al 2015 Antioxidants in heat processed koji and the production mechanisms Food Chem doi 10 1016 foodchem 2015 04 004 Macchi Z et al 2015 A multi center screening trial of rasagiline in patients with amyotrophic lateral sclerosis Possible mitochondrial biomarker target engagement Amyotroph Lateral Scler Frontotemporal Degener 2 1 8 Wada S I et al 2015 Novel autophagy inducers lentztrehaloses A B and C J Antibiot Tokyo doi 10 1038 ja 2015 23 Nishimura T et al 2015 Protective effect of hypotaurine against oxidative stress induced cytotoxicity in rat placental trophoblasts Placenta doi 10 1016 j placenta 2015 02 014 Hutchison A T et al 2014 Black currant nectar reduces muscle damage and inflammation following a bout of high intensity eccentric contractions J Diet Suppl doi 10 3109 1939021 1 2014 952864 Krautbauer S et al 2014 Free fatty acids lipopolysaccharide and IL 1a induce
7. LY Sample extracts for testing 1X PBS and Deionized water 50 Acetone 37 C incubator Bottles flasks and conical or microtubes necessary for reagent preparation Reagents and materials necessary for sample extraction and purification 10 uL to 1000 uL adjustable single channel micropipettes with disposable tips 50 uL to 300 uL adjustable multichannel micropipette with disposable tips Multichannel micropipette reservoir 10 Fluorescence microplate reader equipped with a 480 nm excitation filter and 520 nm emission filter Storage Upon receipt store the Fluorescein Probe 100X Antioxidant Standard and Free Radical Initiator frozen at 20 C and Assay Diluent at 4 C Aliquot as necessary to avoid multiple freeze thaws Store all remaining kit components at room temperature until their expiration dates Preparation of Reagents 1X Assay Diluent Dilute the Assay Diluent 1 4 with deionized water Mix to homogeneity Use this for all sample and standard dilutions Store the 1X Assay Diluent at 4 C 1X Fluorescein Probe Dilute the Fluorescein Probe 1 100 with 1X Assay Diluent Mix to homogeneity Label this as 1X Fluorescein Solution Use only enough Fluorescein Probe as necessary for immediate applications Note Do not store diluted Fluorescein Probe solutions Free Radical Initiator Solution Freshly prepare 80 mg mL Free Radical Initiator Solution in 1X PBS For example weigh out 160 mg of Free Radical Initiator powder in a c
8. adipocyte manganese superoxide dismutase which is increased in visceral adipose tissues of obese rodents PLoS One 9 e86866 Yoo J H et al 2014 Crepidiastrum denticulatum extract protects the liver against chronic alcohol induced damage and fat accumulation in rats J Med Food 17 432 438 Han C H et al 2014 Asn Trp dipeptides improve the oxidative stress and learning dysfunctions in D galactose induced BALB c mice Food Funct 5 2228 2236 Han C H et al 2014 Antioxidant and antiglycation activities of the synthesised dipeptide Asn Trp derived from computer aided simulation of yam dioscorin hydrolysis and its analogue Gln Trp Food Chem 147 195 202 9 CELL BIOLABS INC A A 14 Krautbauer S et al 2014 Manganese superoxide dismutase knock down in 3T3 L1 preadipocytes impairs subsequent adipogenesis Mol Cell Biochem 393 69 76 15 Han C H et al 2014 Effects of yam tuber protein dioscorin on attenuating oxidative status and learning dysfunction in d galactose induced BALB c mice Food Chem Toxicol 65 356 363 16 Yang J et al 2014 Validation of genome wide association study GWAS identified disease risk alleles with patient specific stem cell lines Hum Mol Genet 23 3445 3455 17 Ungvari Z et al 2012 Testing predictions of the oxidative stress hypothesis of aging using a novel invertebrate model of longevity the giant clam Tridacna derasa J Gerontol A Biol Sci Med Sci
9. biomolecules from a variety of samples The ORAC Activity Assay is based on the oxidation of a fluorescent probe by peroxyl radicals by way of a hydrogen atom transfer HAT process Peroxyl radicals are produced by a free radical initiator which quenches the fluorescent probe over time Antioxidants present in the assay work to block the peroxyl radical oxidation of the fluorescent probe until the antioxidant activity in the sample is depleted The remaining peroxyl radicals destroy the fluorescence of the fluorescent probe This assay continues until completion which means both the antioxidant s inhibition time and inhibition percentage of free radical damage is a single value The sample antioxidant capacity correlates to the fluorescence decay curve which is usually represented as the area under the curve AUC The AUC is used to quantify the total peroxyl radical antioxidant activity in a sample and is compared to an antioxidant standard curve of the water soluble vitamin E analog Trolox see Assay Principle below Cell Biolabs OxiSelect ORAC Activity Assay is a fast and reliable kit for the direct measurement of ORAC antioxidant capacity from cell lysate plasma serum tissue homogenates and food extracts This Trial Size kit provides sufficient reagents to perform up to 48 assays including blanks antioxidant standards and unknown samples The assay is designed for use in single plate microplate readers as well as readers with high t
10. e the supernatant as necessary prior to running the assay Certain liquids such as juice extracts may be tested without dilution Preparation of Antioxidant Standard Curve I Hydrophilic aqueous Samples 1 Prepare fresh standards by diluting the 5 mM Antioxidant Standard stock solution to 0 2 mM in Assay Diluent example add 10 uL of Antioxidant Standard stock tube to 240 uL of Assay Diluent 2 Prepare a series of the remaining antioxidant standards according to Table 1 below 5 CELL BIOLABS INC ZA Se abate A 0 2 mM Trolox Resulting Trolox Antioxidant Assay Diluent Concentration Tubes Standard uL uL uM 1 50 150 50 2 40 160 40 3 30 170 30 4 20 180 20 5 10 190 10 6 5 195 5 7 2 5 197 5 2 5 8 0 200 0 Table 1 Preparation of Standards for use when testing Hydrophilic Samples Note Do not store diluted Antioxidant Standard solutions II Lipophilic Samples 1 Prepare fresh standards by diluting the 5 mM Antioxidant Standard stock solution to 0 2 mM in 50 acetone example add 10 uL of Antioxidant Standard stock tube to 240 uL of acetone 2 Prepare a series of the remaining antioxidant standards according to Table 2 below 0 2 mM Trolox Resulting Trolox Antioxidant 50 Acetone Concentration Tubes Standard uL uL uM 1 50 150 50 2 40 160 40 3 30 170 30 4 20 180 20 5 10 190 10 6 5 195 5 T 2 5 197 5 2 5 8 0 200
11. hroughput capabilities Please read the complete kit insert prior to performing the assay 2 CELL BIOLABS INC AS a Assay Principle Blank Negative AUC prank Control Fluorescent AAPH AUC Probe ROO Initiator SSS Antioxidant Standard or Sam ple Sample Integration Net AUC ORAC Capacity AUC AUC Sample Blank Related Products STA 305 OxiSelect Nitrotyrosine Protein ELISA Kit STA 310 OxiSelect Protein Carbonyl ELISA Kit STA 312 OxiSelect Total Glutathione GSSG GSH Assay Kit STA 320 OxiSelect Oxidative DNA Damage ELISA Kit 8 OHdG Quantitation STA 324 OxiSelect Oxidative DNA Damage Quantitation Kit AP Sites STA 330 OxiSelect TBARS Assay Kit MDA Quantitation STA 337 OxiSelect 8 iso Prostaglandin F2a ELISA Kit 96 Assays STA 340 OxiSelect Superoxide Dismutase Activity Assay STA 341 OxiSelect Catalase Activity Assay 10 STA 346 OxiSelect HORAC Activity Assay Ce N DAHA AWN Kit Components 1 96 well Microtiter Plate Part No 234501 One 96 well clear bottom black plate 2 Fluorescein Probe 100X Part No 234502 T One 75 uL vial 3 Free Radical Initiator Part No 234503 One 0 5 g bottle of powder 4 Antioxidant Standard Trolox Part No 234504 One 100 uL vial of a 5 mM solution 5 Assay Diluent 4X Part No 234505 T One 10 mL bottle IN CELL BIOLABS INC Materials Not Supplied eT ONT oe ee
12. mogenize tissue sample on cold PBS and centrifuge at 10 000 x g for 10 minutes at 4 C Aliquot and store the supernatant for use in the assay e Plasma or Serum Dilute 100 fold with Assay Diluent immediately before use e Urine Test neat or diluted with Assay Diluent if appropriate e Nutrition Samples Results may vary depending on sample source and purification Dilution and preparation of these samples is at the discretion of the user but use the following guidelines o Solid or High Protein Samples Weigh solid sample and then homogenize after adding deionized water 1 2 w v Centrifuge the homogenate at 10 12 000 x g for 10 minutes at 4 C Recover the supernatant which is the water soluble fraction Separately recover the insoluble fraction pulp and wash with deionized water Combine this wash with the supernatant The pooled supernatant can be diluted with Assay Diluent and used directly in the assay The pulp is further extracted by adding pure acetone 1 4 w solid pulp v and mixing at room temperature for 30 60 minutes Centrifuge the extract solid at 12 000 x g for 10 minutes at 4 C Recover the acetone extract and dilute with Assay Diluent as necessary prior to running the assay The total ORAC value is calculated by combining the results from the water soluble fraction and the acetone extract from the pulp fraction o Aqueous Samples Centrifuge the sample at 5 10 000 x g for 10 minutes at 4 C to remove any particulates Dilut
13. onical tube and reconstitute the powder with 2 mL of 1X PBS and mix to homogeneity Free Radical Initiator Solution is not stable and should be used immediately Preparation of Samples Note Samples should be stored at 70 C prior to performing the assay Sample should be prepared at the discretion of the user The following recommendations are only guidelines and may be altered to optimize or complement the user s experimental design Deproteinated Fractions Samples can be deproteinated and have their non protein fractions assayed Mix samples with 0 5 M perchloric acid 1 2 v v centrifuge at 10 000 x g for 10 minutes at 4 C Remove the supernatant for measuring the non protein fraction in the assay 4 AN CELL BIOLABS INC S a e Cell Culture Wash cells 3 times with cold PBS prior to lysis Lyse cells with sonication or homogenation in cold PBS and centrifuge at 10 000 x g for 10 minutes at 4 C Aliquot and store the supernatant for use in the assay e Lipophilic Fractions Dissolve lipophilic samples in 100 acetone and then dilute in 50 acetone Incubate the mixture for 1 hour at room temperature with mixing Further dilute samples as necessary prior to testing e Plasma Collect blood with heparin and centrifuge at 4 C for 10 minutes Remove the plasma and aliquot samples for testing Blood plasma or serum should be diluted 100 fold or more with Assay Diluent prior to performing the assay e Tissue Lysate Sonicate or ho
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