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OxiSelect™ Hydroxyl Radical Antioxidant

1. N CELL BIOLABS INC Materials Not Supplied 1 Sample extracts for testing 2 Deionized water 3 37 C incubator 4 Bottles flasks and conical or microtubes necessary for reagent preparation 5 Reagents and materials necessary for sample extraction and purification 6 lO uL to 1000 uL adjustable single channel micropipettes with disposable tips 7 50 uL to 300 uL adjustable multichannel micropipette with disposable tips 8 Multichannel micropipette reservoir 9 Fluorescence microplate reader equipped with a 485 nm excitation filter and 530 nm emission filter Storage Upon receiving store the HORAC Fluorescein Probe 100X and Antioxidant Standard at 20 C and the Fenton Reagent Hydroxyl Radical Initiator and Assay Diluent at 4 C Aliquot as necessary to avoid multiple freeze thaw cycles Store all remaining kit components at room temperature until their expiration dates Preparation of Reagents Assay Diluent 2X Dilute the Assay Diluent 1 2 with deionized water Mix to homogeneity Use this for all sample and standard dilutions Store the 1X Assay Diluent at 4 C Fluorescein Probe 100X Dilute the Fluorescein Probe 1 100 with Assay Diluent Mix to homogeneity Label this as 1X Fluorescein Solution Use only enough Fluorescein Probe as necessary for immediate applications Hydroxyl Radical Initiator 5X Dilute the Hydroxyl Radical Initiator 1 5 with deionized or distilled water Mix to homogeneity Label this so
2. 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 Dilute the supernatant as necessary prior to running the assay CELL BIOLABS INC T nnne Preparation of Antioxidant Standard Curve 1 Prepare fresh standards by diluting the 5 mM Gallic Acid Antioxidant Standard stock solution in 1X Assay Diluent Use only enough Gallic Acid Antioxidant Standard as necessary for immediate applications Prepare a series of the remaining antioxidant standards according to Table 1 below Gallic Acid Gallic Acid Antioxidant Concentration Tubes Standard uL Assay Diluent uL uM 1 90 410 900 2 80 420 800 3 70 430 700 4 60 440 600 5 50 450 500 6 40 460 400 7 30 470 300 8 20 480 200 9 10 490 100 10 0 500 0 Table 1 Preparation of Gallic Acid Antioxidant Standards Note Do not store diluted Antioxidant Standard solutions Prepare fresh Antioxidant Standards for every assay performed Assay Protocol Note Each Antioxidant Standard and samples should be assayed in duplicate or triplicate A freshly prepared standard curve should be used each time the assay is p
3. HORAC Assay is a classic tool for measuring the antioxidant capacity of biomolecules from a variety of samples The HORAC Activity Assay is based on the oxidation of a fluorescent probe by hydroxyl radicals by way of a hydrogen atom transfer HAT process Hydroxyl radicals are produced by hydroxyl radical initiator and fenton reagent which quenches the fluorescent probe over time Antioxidants present in the assay work to block the radical hydroxyl oxidation of the fluorescent probe until the antioxidant activity in the sample is depleted The remaining hydroxyl 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 hydroxyl radical antioxidant activity in a sample and is compared to a gallic acid antioxidant standard curve Cell Biolabs OxiSelect HORAC Activity Assay is a fast and reliable kit for the direct measurement of HORAC antioxidant capacity from cell lysate plasma serum tissue homogenates and food extracts Each Kit provides sufficient reagents to perform up to 192 assays including blanks antioxidant standards and unknown samples The assay is designed for use in single plate microplate readers as
4. 0 50 60 400 600 800 1000 Gallic Acid pM Figure 1 HORAC Activity Assay Standard Curve Calculation of Results Note A spreadsheet application or plate reader software can be used to perform the calculations 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 RFU RFU7 RFU RFUJRFU RFUp relative fluorescence value of time point zero RFUs0 RF Uo RFUqy RFU RFU relative fluorescence value of time points eg RFUs is relative fluorescence value at minute five AUC Blank 10 15 20 25 30 35 40 45 50 55 60 Time min 10 15 20 25 30 35 40 45 50 55 60 AUC Antioxidant Time min j CELL BIOLABS INC 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 Gallic Acid Antioxidant Standard concentration on the x axis see Figure 1 4 Calculate the uMole Gallic Acid Equivalents GAE of unknown sample by comparing the standard curve Results HORAC value may be expressed as GAE per L or g of sample Calculation Example 20 uL of 10 fold diluted sample is assayed along with 20 uL of each Gallic Acid antioxidant s
5. 015 Cell Biolabs Inc All rights reserved No part of these works may be reproduced in any form without permissions in writing 10 CELL BIOLABS INC P
6. NOTE Revisions to Preparation of Reagents Product Manual OxiSelect Hydroxyl Radical Antioxidant Capacity HORAC Activity Assay Catalog Number STA 346 192 assays STA 346 5 5 x 192 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 Hydroxyl Radical Antioxidant Capacity
7. erformed 1 Add 20 uL of the diluted Antioxidant Standards or samples to be tested to the 96 well Microtiter Plate Add 140 uL of the 1X Fluorescein Solution to each well Mix thoroughly Incubate the plate for 30 minutes at room temperature Add 20 uL of the 1X Hydroxyl Radical Initiator into each well using either a multichannel pipette or a plate reader liquid handling system Immediately add 20 uL of the Fenton Reagent to each well Shake the plate immediately for 15 seconds to ensure homogeneity Immediately begin reading sample and standard wells with a fluorescent microplate reader with an excitation wavelength of 480nm and an emission wavelength of 530nm 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 AN CELL BIOLABS INC A eS a Example of Results The following figure demonstrates typical OxiSelect HORAC Activity Assay results One should use the data below for reference only This data should not be used to interpret or calculate actual sample results 1400 1200 1000 800 4 m Blank 800 pM Gallic Acid RFU 600 Net AUC 400 200 no A O N O Time Min 0 10 20 30 4
8. lution as 1X Hydroxyl Radical Initiator Solution Use only enough Hydroxyl Radical Initiator as necessary for immediate use Note Do not store diluted Fluorescein Probe or Hydroxyl Radical Initiator solutions 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 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 4 les CELL BIOLABS INC p Lipophilic Fractions Lipophilic samples must be treated in order to be soluble in an aqueous environment Dissolve samples in 100 acetone and then dilute in a solution of 7 beta cyclodextrin and 5096 acetone Incubate the mixture for 1 hour at room temperature with mixing Further dilute samples as necessary prior to testing Plasma Collect blood with heparin and centrifuge at 4 C for 10 minutes Remove the plasma and aliquot
9. n in patients with symptomatic peripheral artery disease J Vasc Surg 61 1249 1257 Gardner A W et al 2014 Greater endothelial apoptosis and oxidative stress in patients with peripheral artery disease Int J Vasc Med doi 10 1155 2014 160534 Gardner A W et al 2014 Impaired vascular endothelial growth factor A and inflammation in patients with peripheral artery disease Angiology 65 683 690 Jeong M H et al 2014 Protective activity of a novel resveratrol analogue HS 1793 against DNA damage in 137Cs irradiated CHO K1 cells J Radiat Res 55 464 475 Bailey Downs et al 2013 Aging exacerbates obesity induced oxidative stress and inflammation in perivascular adipose tissue in mice a paracrine mechanism contributing to vascular redox dysregulation and inflammation J Gerontol A Biol Sci Med Sci 68 780 792 Ungvari Z et al 2013 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 68 359 367 Downs L C et al 2012 Aging exacerbates obesity induced oxidative stress and inflammation in perivascular adipose tissue in mice a paracrine mechanism contributing to vascular redox dysregulation and inflammation J Gerontol A Biol Sci Med Sci 10 1093 gerona gls238 Bailey Downs L C et al 2011 Liver specific knockdown of IGF 1 decreases vascular oxidative stress resistance by impairing the Nrf2 dependen
10. samples for testing Blood plasma or serum should be diluted 100 fold or more with Assay Diluent prior to performing the assay Tissue Lysate Sonicate or homogenize 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 Plasma or Serum Once samples are collected they may be tested after diluted 100 fold with Assay Diluent Urine Once samples are collected they may be tested directly or diluted with Assay Diluent if appropriate Nutrition Samples Nutritional sample results may vary depending on sample source and purification Dilution and preparation of these samples is at the discretion of the user Aqueous samples such as juice extracts may be tested directly without further manipulation Solid food samples or high protein samples may be processed as follows o Solid 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 The solid pulp or insoluble fraction is also recovered and washed 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
11. t antioxidant response a novel model of vascular aging J Gerontol A Biol Sci Med Sci 67A 313 329 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 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 9 CELL BIOLABS INC T nnnm Contact Information Cell Biolabs Inc 7758 Arjons Drive San Diego CA 92126 Worldwide 1 858 271 6500 USA Toll Free 1 888 CBL 0505 E mail tech cellbiolabs com www cellbiolabs com 2008 2
12. tandard including blank as described in Assay Protocol The average AUC is 2 2 for blank and 7 0 for sample Net AUC AUC Antioxidant AUC blank 7 0 2 2 4 8 Based on the Gallic Acid antioxidant standard curve the equivalent Gallic Acid concentration is 400 uM therefore HORAC value Sample 400 uM x 10 dilution factor 4000 uM GAE 4000 uMole GAE L References 1 Huang D Ou B amp Prior R 2005 J Agric Food Chem 53 1841 1856 2 Ou B Hampsch Woodill M and Prior R 2001 J Agric Food Chem 49 4619 4626 3 Ou B Hampsch Woodill M Flanagan J Deemer E Prior R and Huang D 2002 J Agric Food Chem 50 2772 2777 CELL BIOLABS INC Pp acc Recent Product Citations l 10 11 12 13 Gardner A W et al 2015 Endothelial cell inflammation and antioxidant capacity are associated with exercise performance and microcirculation in patients with symptomatic peripheral artery disease Angiology doi 10 1177 0003319714566863 Jeong M H et al 2014 In vitro evaluation of Cordyceps militaris as a potential radioprotective agent Int J Mol Med 34 1349 1357 Mishra S et al 2014 Semiquinone glucoside derivative SQGD isolated from Bacillus sp INM 1 protects against gamma radiation induced oxidative stress Environ Toxicol Pharmacol 37 553 562 Gardner A W et al 2014 Gender and racial differences in endothelial oxidative stress and inflammatio
13. well as readers with high throughput capabilities Please read the complete kit insert prior to performing the assay 2 las CELL BIOLABS INC AO Assay Principle Blank Negative Control Fluorescent Probe OH Initiator M I Antioxidant Standard or Sample Integration Net AUC HORAC Capacity AUC AUC Sample Blank im AUC ple Related Products STA 305 OxiSelect M Nitrotyrosine Protein ELISA Kit STA 310 OxiSelect M 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 345 OxiSelect ORAC Activity Assay CoN DAHA AR WN c Kit Components 96 well Microtiter Plate Part No 234501 Two 96 well clear bottom black plates HORAC Fluorescein Probe 100X Part No 234601 One 0 5 mL tube Hydroxyl Radical Initiator 5X Part No 234602 One 1 0 mL amber tube Gallic Acid Antioxidant Standard Part No 234603 One 1 5 mL tube of a 5 mM solution Fenton Reagent Part No 234604 One 5 0 mL bottle Assay Diluent 2X Part No 234605 One 50 mL bottle pe ur ue cu ee x

OxiSelect™ Hydroxyl Radical Antioxidant

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OxiSelect™ Hydroxyl Radical Antioxidant