OxiSelect™ 96-Well Comet Assay Kit
Contents
1. Product Manual OxiSelect 96 Well Comet Assay Kit Catalog Number STA 355 96 assays STA 355 5 5 x 96 assays FOR RESEARCH USE ONLY Not for use in diagnostic procedures CELL BIOLABS INC Creating Solutions for Life Science Research Introduction DNA damage due to environmental factors and normal metabolic processes inside the cell occurs at a rate of 1 000 to 1 000 000 molecular lesions per cell per day While this counts for only a small part of the human genome s approximately 6 billion bases 3 billion base pairs unrepaired lesions to critical genes can impede a cell s ability to carry out its function and appreciably increase the likelihood of cancer The comet assay or single cell gel electrophoresis assay SCGE is a common technique for measurement of DNA damage in individual cells Under an electrophoretic field damaged cellular DNA containing fragments and strand breaks is separated from intact DNA yielding a classic comet tail shape under the microscope Extent of DNA damage is usually visually estimated by comet tail measurement however image analysis software is also available for measuring various parameters The OxiSelect 96 Well Comet Assay is a fast and sensitive kit for the measurement of cellular DNA damage Each kit provides sufficient reagents to perform up to 96 assays Assay Principle Cell Biolabs OxiSelect 96 Well Comet Assay is a single cell gel electrophoresis assay SCGE for2. simple evaluation of cellular DNA damage First individual cells are mixed with molten agarose before application to the OxiSelect 96 Well Comet Slide These embedded cells are then treated with a lysis buffer and alkaline solution which relaxes and denatures the DNA Finally the samples are electrophoresed in a horizontal chamber to separate intact DNA from damaged fragments Following electrophoresis the samples are dried stained with a DNA dye and visualized by epifluorescence microscopy Under these conditions the damaged DNA containing cleavage and strand breaks will migrate further than intact DNA and produce a comet tail shape see Figure 1 Each kit provides sufficient reagents to perform up to 96 assays CELL BIOLABS INC Combine cells with OxiSelect Comet Agarose at 37 C Pipette cells onto the OxiSelect 96 Well Comet Slide Treat cells with Lysis Buffer and Alkaline Solution Perform electrophoresis under alkaline or neutral conditions Stain cells with DNA Dye Healthy Cells Damaged Cells Figure 1 Assay Principle A CELL BIOLABS INC _ EN ERAT j Related Products Se SS ee ee a STA 320 OxiSelect Oxidative DNA Damage ELISA Kit 8 OHdG Quantitation STA 321 OxiSelect DNA Double Strand Break DSB Staining Kit STA 324 OxiSelect Oxidative DNA Damage Quantitation Kit AP sites STA 325 OxiSelect Oxidative RNA Damage ELISA Kit 8 OHG Quantitation STA 350
3. C in a water bath for 20 minutes or until agarose liquefies Transfer the bottle to a 37 C water bath for 20 minutes and maintain until needed Vista Green DNA Dye Prepare a 1X Vista Green DNA Staining Solution by diluting the provided stock 1 10000 in TE Buffer 10 mM Tris pH 7 5 1 mM EDTA The solution can be stored at 4 C for up to 3 weeks protected from light Lysis Buffer To prepare 100 mL of 1X Lysis Buffer NaCl 14 6 g EDTA Solution provided 20 0 mL 10X Lysis Solution provided 10 0 mL DMSO 10 0 mL optional for heme containing samples DI H2O Adjust volume to 90 mL Mix thoroughly to dissolve NaCl Slowly adjust the Lysis Buffer to pH 10 0 with 10 N NaOH then QS to 100 mL with DI H20 Chill Lysis Buffer to 4 C before use Note Buffer will appear cloudy at room temperature but will clear at 4 C pH will also remain 10 0 Alkaline Solution To prepare 100 mL of Alkaline Solution NaOH 1 2g EDTA Solution provided 0 2 mL DI H2O Adjust volume to 100 mL Mix thoroughly to dissolve NaOH Chill Alkaline Solution to 4 C before use Electrophoresis Running Solution Choose the appropriate electrophoresis solution based on the desired running conditions and assay sensitivity TBE is preferred for analysis of apoptosis and enables use of the tail length rather than the tail moment for data analysis TBE electrophoresis will detect single stranded and double stranded DNA breaks and may detect a few AP sites Alkali
4. Comet Assay analysis software programs are commercially available such as Comet Assay IV Perceptive Instruments and CASPlab Tail Moment Length is measured from the center of the head to the center of the tail see Figure 3 1 Ostling O and Johanson K J 1984 Micro gel electrophoretic study of radiation induced DNA damages in individual mammalian cells Biochem Biophys Res Commun 123 291 298 2 Singh N P McCoy M T Tice R R and Schneider E L 1988 A simple technique for quantification of low levels of DNA damage in individual cells Exp Cell Res 175 184 191 3 Olive P L Banath J P and Durand R E 1990a Heterogeneity in radiation induced DNA damage and repair in tumor and normal cells using the Comet assay Radiat Res 122 86 94 4 De Boeck M Touil N De Visscher G Vande P A and Kirsch Volders M 2000 Validation and implementation of an internal standard in Comet assay Mutat Res 469 181 197 Recent Product Citations 1 Jones D A et al 2014 Changes in markers of oxidative stress and DNA damage in human visceral adipose tissue from subjects with obesity and type 2 diabetes Diabetes Res Clin Pract 106 627 633 jN CELL BIOLABS INC P 2 Aydin E et al 2014 The effect of carvacrol on healthy neurons and N2a cancer cells some biochemical anticancerogenicity and genotoxicity studies Cytotechnology 66 149 157 3 Tyagi A et al 2011 Re
5. OxiSelect Comet Assay Kit 3 Well Slides 15 Assays STA 351 OxiSelect Comet Assay Kit 3 Well Slides 75 Assays STA 352 OxiSelect Comet Assay Slides 3 Well 5 Slides STA 353 OxiSelect Comet Assay Slides 3 Well 25 Slides STA 354 OxiSelect Comet Assay Control Cells 10 STA 356 OxiSelect 96 Well Comet Assay Slide Kit Components 1 ae YS OxiSelect 96 Well Comet Slide Part No STA 356 One slide OxiSelect Comet Agarose Part No 235002 One sterile 15 mL bottle Vista Green DNA Dye 10000X Part No 235003 One 5 uL vial EDTA Solution 500 mM Part No 235004 One 50 mL bottle 10X Lysis Solution Part No 235005 One 20 mL bottle Materials Not Supplied oe a oe SP e W N NaCl powder NaOH pellets 10 N NaOH for pH adjustment DMSO optional 70 Ethanol TE Buffer 10 mM Tris pH 7 5 1 mM EDTA PBS without Mg and Ca and DI H2O EDTA disodium salt 37 C and boiling water baths Horizontal electrophoresis chamber Adjustable single channel micropipettes with disposable tips Adjustable multichannel micropipette with disposable tips Epifluorescence microscope with FITC filter CELL BIOLABS INC Storage Upon receipt store the Vista Green DNA Dye at 20 C Store all other kit components at room temperature until their expiration dates Preparation of Reagents OxiSelect Comet Agarose Heat the Comet Agarose bottle at 90 95
6. agents Incubate at room temperature for 15 minutes 7 View slides by epifluorescence microscopy using a FITC filter Example of Results The following figures demonstrate typical OxiSelect 96 Well Comet Assay Kit results One should use the data below for reference only This data should not be used to interpret actual results Figure 2 Etoposide Treatment of Jurkat Cells Jurkat cells were untreated left or treated right with 20 uM Etoposide for 4 hours before performing Comet Assay alkaline electrophoresis conditions 33 V 300 mA for 15 minutes 8 fr CELL BIOLABS INC a Calculation of Results The DNA damage is quantified by measuring the displacement between the genetic material of the nucleus comet head and the resulting tail Tail Moment and Tail DNA are the two most common parameters to analyze Comet assay results At least 50 100 cells should be analyzed per sample The Tail Moment has been suggested to be an appropriate index of induced DNA damage in considering both the migration of the genetic material as well as the relative amount of DNA in the tail Tail Moment Length Figure 3 Typical Damaged DNA in Comet Assay References Tail DNA 100 x Tail DNA Intensity Cell DNA Intensity Tail Moment can be measured using one of the following methods a Olive Tail Moment Tail DNA x Tail Moment Length b Extent Tail Moment Tail DNA x Length of Tail see Figure 3 A number of
7. ain suspensions at 37 C to avoid gelation Titrate samples again Just prior to slide addition 6 Maintaining the slide horizontally transfer the slide to 4 C in the dark for 15 minutes 7 Carefully transfer the slide to a small basin container containing pre chilled Lysis Buffer 50 100 mL slide Immerse the slide in the buffer for 30 60 minutes at 4 C in the dark 8 Carefully aspirate the Lysis Buffer from the container and replace with pre chilled Alkaline Solution 50 100 mL slide Immerse the slide in the solution for 30 minutes at 4 C in the dark Assay Protocol I TBE Electrophoresis 1 Aspirate the Alkaline Solution from the container and replace with pre chilled TBE Electrophoresis Solution Immerse the slide for 5 minutes and then repeat once more Maintaining the slide horizontally carefully transfer the slide to a horizontal electrophoresis chamber Fill the chamber with cold TBE Electrophoresis Solution until the buffer level covers the slide Apply voltage to the chamber for 10 15 minutes at 1 volt cm e g if the chamber electrodes are 35 cm apart you would then apply 35 volts to the slide Maintaining the slide horizontally carefully transfer the slide from the electrophoresis chamber to a clean small basin container containing pre chilled DI H2O 50 100 mL slide Immerse the slide for 2 minutes aspirate and then repeat twice more 5 Aspirate the final water rinse and replace with cold 70 Ethanol
8. for 5 minutes 6 Maintaining the slide horizontally remove the slide from the 70 Ethanol and allow to air dry Once the agarose and slide is completely dry add 50 uL well of diluted Vista Green DNA Dye see Preparation of Reagents Incubate at room temperature for 15 minutes View slides by epifluorescence microscopy using a FITC filter II Alkaline Electrophoresis 1 Maintaining the slide horizontally carefully transfer the slide from the Alkaline Solution to a horizontal electrophoresis chamber Fill the chamber with cold Alkaline Electrophoresis Solution until the buffer level covers the slide Apply voltage to the chamber for 15 30 minutes at 1 volt cm e g if the chamber electrodes are 35 cm apart you would then apply 35 volts to the slide Additionally adjust the volume of Alkaline Electrophoresis Solution to produce a current setting of 300 mA Maintaining the slide horizontally carefully transfer the slide from the electrophoresis chamber to a clean small basin container containing pre chilled DI H2O 50 100 mL slide Immerse the slide for 2 minutes aspirate and then repeat twice more Aspirate the final water rinse and replace with cold 70 Ethanol for 5 minutes 5 Maintaining the slide horizontally remove the slide from the 70 Ethanol and allow to air dry o gt CELL BIOLABS INC 6 Once the agarose and slide is completely dry add 50 uL well of diluted Vista Green DNA Dye see Preparation of Re
9. ls at 700 x g for 2 minutes and discard supernatant Wash cell pellet once with ice cold PBS without Mg and Ca centrifuge and discard the supernatant Finally resuspend the cells at 1 x 10 cells mL in ice cold PBS without Mg and Ca Adherent Cells Gently remove cells from flask dish by scraping with a rubber policeman Transfer cell suspension to a conical tube and centrifuge at 700 x g for 2 minutes discarding the supernatant Wash cell pellet once with ice cold PBS without Mg and Ca centrifuge and discard the supernatant Finally resuspend the cells at 1 x 10 cells mL in ice cold PBS without Mg and Ca Tissue Preparation Using dissection scissors mince a small piece of tissue in 1 2 mL of ice cold PBS containing 20 mM EDTA without Mg and Ca Allow the tissue cell suspension to stand for 5 minutes before transferring the supernatant to a centrifuge tube avoid transferring debris Centrifuge discarding the supernatant and then resuspend the cells at 1 x 10 cells mL in ice cold PBS without Mg and Ca In a pre warmed 96 well plate combine cell samples with Comet Agarose step 2 at 1 10 ratio v v Titrate to mix and immediately pipette 20 uL well onto the OxiSelect 96 Well Comet Slide using a multichannel micropipette Ensure complete well coverage by spreading the suspension over the well with the pipette tip CELL BIOLABS INC am Note For multiple samples maint
10. ne electrophoresis is more sensitive and will detect smaller amounts of DNA damage Alkaline electrophoresis will detect single stranded and double stranded DNA breaks the majority of AP sites and alkali labile DNA adducts To prepare 1 L of Electrophoresis Solution 1 TBE Electrophoresis Solution Tris Base 10 8 g CELL BIOLABS INC P o gt Boric Acid 5 5g EDTA disodium salt 0 93 g DI H2O Adjust volume to 1 L Mix thoroughly to dissolve solids Chill TBE Running Solution to 4 C before use OR 2 Alkaline Electrophoresis Solution 300 mM NaOH pH gt 13 1 mM EDTA NaOH 12 0 g EDTA Solution provided 2 0 mL DI H2O Adjust volume to 1 L Mix thoroughly to dissolve NaOH Chill Alkaline Running Solution to 4 C before use Special Precautions To avoid ultraviolet light damage to cell samples perform the assay under low dim light conditions Preparation of Samples and Slides 1 Prepare Lysis Buffer Alkaline Solution and Electrophoresis Running Solution see Preparation of Reagents prior to performing the assay Chill all solutions to 4 C thoroughly Heat OxiSelect Comet Agarose to 90 95 C in a water bath for 20 minutes or until agarose liquefies Cool the agarose by transferring the bottle to a 37 C water bath for 20 minutes Heat OxiSelect 96 Well Comet Slide at 37 C for 15 minutes to promote agarose attachment Prepare cell samples including controls as follows Suspension Cells Centrifuge cel
11. sveratrol selectively induces DNA Damage independent of Smad4 expression in its efficacy against human head and neck squamous cell carcinoma Clin Cancer Res 17 5402 5411 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 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 2015 Cell Biolabs Inc All rights reserved No part of these works may be reproduced in any form without permissions in writing 10 eo CELL BIOLABS INC L AU
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