DNA Damage & Repair Reagents

T4-Endo V FLARE™ Assay Kit

Catalog Number 4055-100-FK

T4-Endo V FLARE Assay Kit

Catalog Number: 4055-100-FK

Reagent Kit for the Analysis of DNA Damage in SingleCells Using the CometAssay™ and T4-Endo V.

This package insert must be read in its entirety before using this product.

FOR RESEARCH USE ONLY.NOT FOR USE IN DIAGNOSTIC PROCEDURES.

TABLE OF CONTENTSContents Page

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2REAGENTS PROVIDED 3ADDITIONAL REAGENTS AND MATERIALS REQUIRED . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3PROCEDURE 4

Reagent Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4Sample Preparation 6Cell Preparation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6Tissue Preparation 7Method for Cryopreservation of Cells Prior to FLARE Assay . . . . . . . . . . . . . . . . . . . . . . . . . . 7

FLARE ASSAY PROTOCOL 7CONTROLS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9DATA ANALYSIS 10TROUBLESHOOTING GUIDE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11WARNINGS 12APPENDICES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

A. Reagents and Buffer Composition 12B. Suggestions for Assay Optimization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

REFERENCES 14

MANUFACTURED FOR AND DISTRIBUTED BY:R&D Systems, Inc. TELEPHONE: (800) 343-7475614 McKinley Place NE (612) 379-2956Minneapolis, MN 55413 FAX: (612) 656-4400United States of America E-MAIL: info@RnDSystems.com

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R&D Systems Europe77 boulevard Vauban FREEPHONE: +0800 90 72 4959800 LILLE FAX: +0800 77 16 68France E-MAIL: info@RnDSystems.co.uk

INTRODUCTIONThe T4-Endo V FLARE (Fragment Length Analysis using Repair Enzymes) Assay Kit providesthe reagents to screen for DNA damage in single cells using T4-Endo V (T4-Pyrimide DimerGlycosylase/T4-PDG) in conjunction with the CometAssay™ single cell gel electrophoresis kit.To assess the type of DNA damage by a putative mutagen, drug, or treatment regimen, cellsare harvested after treatment and immobilized in a layer of low melting point agarose on theFLARE Slide. The cells are gently lysed and then incubated with T4-Endo V, which recognizescis-syn cyclobutane pyrimidine dimers and AP sites. T4-Endo V also cleaves AP sites on bothdouble and single-stranded DNA.

The slides are then immersed in an alkali solution to unwind the DNA strands, followed byneutral gel electrophoresis. The denatured, cleaved DNA fragments migrate out of the cellunder the influence of an electric field, whereas undamaged supercoiled DNA remains withinthe confines of the nuclear cell membrane. Evaluation of the DNA “comet” tail shape andmigration pattern after staining with a fluorescent DNA intercalating dye or silver stainingallows for assessment of the extent of DNA damage. The type of DNA damage is inferred fromthe substrate specificity of T4-Endo V.

Qualitative data may be generated if the comets are scored according to categories of small tolarge comet tail DNA content. Quantitative and statistical data can readily be generated byanalysis of the results with image analysis software that can calculate tail length and tailmoment (see Data Analysis section).

The FLARE Slide promotes adherence of low melting point agarose, eliminating the timeconsuming and unreliable traditional method of preparing base layers of agarose. These slidesshorten assay time and permit the rapid and reliable analysis of large numbers of samples in astandard format. SYBR� Green I is included in the kit for DNA visualization with improvedsensitivity compared to ethidium bromide.

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REAGENTS PROVIDED

Component Amount Provided Storage Temperature

T4-Endo V 100,000 Units 2 - 8° C

25X FLARE Buffer 1 40 mL Room temperature

REC™ Dilution Buffer 10 mL � -20° C in a manual defrost freezer

100X BSA Additive 100 �L � -20° C in a manual defrost freezer

FLARE Slides 25 each* Room temperature

Lysis Solution 2 x 500 mL Room temperature

Comet LMAgarose 15 mL Room temperature

SYBR Green Inucleic acid gel stain

5 �L � -20° C in a manual defrost freezer

*Additional FLARE Slides (100 each), Catalog # 3950-300-02.

ADDITIONAL REAGENTS AND MATERIALS REQUIRED

Reagents� 10X PBS, Ca2+ and Mg2+ free.� NaOH pellets.� Dimethylsulfoxide (optional).� Ethanol.� Glycerol.� 500 mM EDTA, pH 8.0.� TE Buffer (10 mM Tris-HCl, pH 7.5; 1 mM EDTA).� p-Phenylenediamine dihydrochloride.� 10X TBE Buffer (see Appendix A, page 12)� Deionized water.� 3M NaCl

Equipment� Adjustable pipettes and pipette tips (1 - 20 �L, 20 - 200 �L, 200 - 1000 �L pipettors).� Serological pipettes.� Boiling water bath and 37° C water bath.� Horizontal electrophoresis apparatus.� Epifluorescence microscope equipped with a Fluorescein filter.� 2 liter beaker.� 2 - 8° C refrigerator.� Humidity chamber.

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PROCEDUREReagent PreparationReagents marked with an asterisk (*) should be prepared immediately before use. Weargloves, lab coat and eye protection when handling any chemical reagents. The volumesgiven for each reagent are based on processing samples of up to 4 cm2, immobilized on glassslides. Different configurations of chamber slides, culture plates, free floating sections and theuse of glass coverslips may require adjustments to the stated volumes.

1. 1X PBS, Ca2+ and Mg2+ freeDilute 10X PBS with deionized water to prepare 1X PBS. Store at room temperature.

2. Lysis SolutionFor up to 10 slides (3 samples per slide) prepare:

Lysis Solution 40 mL

DMSO 4 mL (optional)

Chill at 2 - 8° C, or on ice, for at least 20 minutes before use. The addition of DMSO isoptional and is required only for samples containing heme, such as blood cells or tissuesamples.

3. Comet LMAgaroseThe Comet LMAgarose is ready to use once molten. Loosen the cap to allow forexpansion, then heat the bottle in a boiling water bath for 5 minutes or until the agarose ismolten. (Caution: Microwaving is not recommended). Place the bottle in a 37° C waterbath for 10 minutes to cool. The LMAgarose will remain molten indefinitely at 37° C.

4. Alkali Solution, pH 12.1*Prepare on the day of use. Standardizing the pH meter with pH 10.0 standard bufferimmediately before making the Alkali Solution is recommended. In a 2 liter beaker,combine:

3M NaCl 200 mL

500 mM EDTA, pH 8.0 4 mL

Deionized water 1700 mL

Add 5 N NaOH dropwise with stirring until the pH of the solution reaches 12.1. Adddeionized H2O to a final volume of 2 liters. Store 100 mL (if using 1 Coplin jar) at roomtemperature and store the remainder at 2 - 8° C.

5. 1X TBE BufferDilute the 10X TBE (see Appendix A) to 1X in deionized water to prepare working strengthbuffer.

6. Initial T4-Endo V Dilutions*The final concentration of T4-Endo V to be used in the FLARE assay must be optimized foryour particular cell line and type of exposure to maximize the difference in comet sizebetween experimental samples treated with T4-Endo V and those exposed to reactionbuffer only. Typical final dilutions for T4-Endo V are 1:100, 1:500, and 1:1,000. Werecommend making initial dilutions (e.g. 1:5 and 1:10) in REC Dilution Buffer. The longterm stability of diluted T4-Endo V has not been established, therefore, immediate use ofthe diluted enzyme is recommended.

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7. 1X FLARE Buffer 1Prepare 150 mL of 1X FLARE Buffer 1 (sufficient for 10 slides):

25X FLARE Buffer 1 6 mL

Deionized water 144 mL

8. T4-Endo V FLARE Reaction BufferPrepare 1 mL of T4-Endo V FLARE Reaction Buffer (sufficient for 10 samples):

25X FLARE Buffer 1 40 �L

100X BSA Additive 10 �L

Deionized water 950 �L

Store at 2 - 8° C for up to 1 week or freeze at � -20° C in a manual defrost freezer.

9. Working T4-Endo V Enzyme Solutions*Each sample area on the FLARE Slide requires 100 �L of working T4-Endo V EnzymeSolution prepared as follows (for 1 sample area):

T4-Endo V FLARE Reaction Buffer (from step 8) 100 �L

T4-Endo V Enzyme, undiluted or diluted (from step 6) 1 �L

Place on ice and use immediately.

10. SYBR Green I Staining SolutionPrepare SYBR Green I Staining Solution from the SYBR Green I nucleic acid gel stainprovided (10,000X concentrate in DMSO).

SYBR Green I nucleic acid gel stain 1 �L

TE Buffer, pH 7.5(TE: 10 mM Tris-Cl (pH 7.5), 1mM EDTA)

10 mL

The diluted stock is stable for several weeks when stored at 2 - 8° C in the dark.

11. Anti-fade SolutionPrepare if fading of samples occurs. In a 50 mL tube, mix until dissolved:

p-Phenylenediamine dihydrochloride 500 mg

1X PBS 4.5 mL

Adjust the pH to 7.5 - 8.0 with dropwise addition of 10 N NaOH. Add 1X PBS to increasethe volume to 5 mL, then add 45 mL of glycerol for a final volume of 50 mL. Vortex themixture thoroughly and apply 10 �L per sample, covering samples with coverslips. Nailpolish may be used to seal the coverslip. Re-staining of slides is not recommended. Storeanti-fade solution at � -20° C in a manual defrost freezer up to 1 month. Darkening of thesolution may occur.

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Sample Preparation

Cell samples should be prepared immediately before starting the assay, although success hasbeen obtained using cryopreserved cells (see below). Cell samples should be handled underdimmed or yellow light to prevent DNA damage from ultraviolet light. Buffers should be chilledto 2 - 8° C or kept on ice to inhibit endogenous damage occurring during sample preparationand to inhibit repair in the unfixed cells. PBS must be calcium and magnesium free to inhibitendonuclease activity. The appropriate controls should also be included (see below). Optimalresults are usually obtained with 500 - 1000 cells per FLARE Slide sample area. Using 50 �Lof a cell suspension at 1 x 105 cells per mL combined with 500 �L of LMAgarose will providethe correct agarose concentration and cell density for optimal results when plating 75 �L persample.

Cell Preparation

Note: 1X PBS must be Ca2+ and Mg2+ free.

Suspension CellsCell suspensions are harvested by centrifugation. Resuspend cells at 1 x 105 cells/mL inice cold 1X PBS. The media used for cell culture can reduce the adhesion of the agaroseon the Comet Slides.

Adherent CellsTrypsinize cells with Trypsin-EDTA (0.25% Trypsin, 1 mM EDTA). First, wash themonolayer of cells with sterile 1X PBS, warmed to 37° C. Add enough Trypsin-EDTA tocoat the entire monolayer of cells. Incubate the flask for 2 minutes at 37° C or until cellseasily detach upon tapping of the flask. Add 10 mL of complete media (containing fetalbovine serum) to inactivate the trypsin. Transfer the cells into a centrifuge tube. Performcell count and then pellet cells. Wash once in ice cold 1X PBS. Resuspend at1 x 105 cells/mL in ice cold 1X PBS.

Tissue Preparation

Note: 1X PBS must be Ca2+ and Mg2+ free.

Place a small piece of tissue into 1 - 2 mL of ice cold 20 mM EDTA in 1X PBS. Using smalldissecting scissors, mince the tissue into very small pieces and let stand for 5 minutes.Recover the cell suspension, avoiding transfer of debris. Count cells, pellet bycentrifugation, and resuspend at 1 x 105 cells/mL in ice cold 1X PBS.

For blood rich organs (e.g., liver, spleen), chop the tissue into large pieces (1 - 2 mm3), letsettle for 5 minutes then aspirate and discard medium. Add 1 - 2 mL of ice cold 20 mMEDTA in 1X PBS, mince the tissue into very small pieces and let stand for 5 minutes.Recover the cell suspension, avoiding transfer of debris. Count cells, pellet, andresuspend at 1 x 105 cells/mL in ice cold 1X PBS.

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Method for Cryopreservation of Cells Prior to FLARE Assay

Note: 1X PBS must be Ca2+ and Mg2+ free.

Certain cells (e.g. lymphocytes) may be successfully cryopreserved prior to performing theCometAssay (see reference 7). A pilot study should be performed to determine ifcryopreservation is appropriate for the cells in use.

1. Centrifuge cells at 200 x g for 5 minutes.

2. Resuspend the cell pellet at 1 x 107 cells/mL in 10% (v/v) dimethylsulfoxide, 40%(v/v) medium, 50% (v/v) fetal calf serum.

3. Transfer aliquots of 2 x 106 cells into freezing vials.

4. Freeze at � -70° C with -1° C per minute freezing rate.

5. Recover cells by submerging in a 37° C water bath until the last trace of ice hasmelted.

6. Transfer to 15 mL of prechilled 40% (v/v) medium, 10% (w/v) dextrose, 50% (v/v) fetalcalf serum.

7. Centrifuge at 200 x g for 10 minutes at 2 - 8° C.

8. Resuspend in ice cold 1X PBS and proceed with FLARE Assay.

FLARE ASSAY PROTOCOLModifications may be necessary for use of the T4-Endo V FLARE assay kit with other types ofslides. The protocol utilizes neutral electrophoresis conditions to detect single stranded DNAbreaks, double-stranded DNA breaks, apurinic sites, and apyrimidinic sites. Prior to performingthe FLARE assay, a cell viability assay should be performed to determine the dose of the testsubstance that gives at least 75% viability. False positives may occur when high doses ofcytotoxic agents are used.

The FLARE assay requires approximately 3 hours to complete. Once the cells or tissues havebeen prepared, the procedure is not labor intensive. The Lysis Solution can be chilled and theLMAgarose melted while the cell and tissue samples are being prepared.

All steps are performed at room temperature (18 - 24° C) unless otherwise specified.Work under dimmed or yellow light to prevent damage from UV.

1. Prepare Lysis Solution (Reagent Preparation, step 2).

2. Melt LMAgarose (Reagent Preparation, step 3).

3. Refer to the Sample Preparation section. Combine cells at 1 x 105/mL with moltenLMAgarose (37° C) at a ratio of 1:10 (v/v) and immediately pipette 75 �L onto the FLARESlide. Use the side of the pipette tip to spread the agarose/cells over the sample area.

Comet LMAgarose 500 �L

Cells in 1X PBS (Ca2+ and Mg2+ free) 50 �L

Note: If sample is not spreading evenly on the slide, warm the slide to 37° C beforeapplication.

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4. Place the FLARE slide flat at 2 - 8° C in the dark (in a refrigerator) for 10 minutes.A 0.5 mm clear ring will appear at edge of the sample area. Increasing gelling time to30 minutes improves adherence of samples in high humidity environments.

5. Immerse the slide in prechilled Lysis Solution and leave on ice or at 2 - 8° C for30 - 60 minutes.

6. Tap off excess buffer from the slide and immerse in freshly prepared 1X FLARE Buffer 1(Reagent Preparation, step 7) at room temperature to equilibrate the sample. Change the1X FLARE Buffer 1 solution three times over a 15 minute period.

7. Add 100 �L of working T4-Endo V Enzyme Solution (Reagent Preparation, step 9) to eachsample area. Remember to include a buffer-only control. Carefully place slides in ahumidity chamber and incubate at 37° C for 30 - 60 minutes. Example: For sample area 1on a FLARE slide, apply buffer only; sample area 2, 1:500 dilution of T4-Endo V; andsample area 3, 1:1000 dilution of T4-Endo V.

8. Transfer the FLARE Slide to a Coplin jar containing Alkali Solution at pH 12.1 (ReagentPreparation, step 4) and incubate for 30 minutes at room temperature in the dark. Changethe Alkali Solution once.

9. Rinse slides in a 1X TBE (Reagent Preparation, step 5) in the Coplin Jar. Transfer slides toa horizontal electrophoresis apparatus. Place the slides flat onto a gel tray and alignequidistant from the electrodes. Carefully pour 1X TBE Buffer until the level just covers theslides. Set the voltage to about 1 Volt/cm. Perform electrophoresis for 20 - 40 minutes atroom temperature.

Note: Alkali electrophoresis may be performed for higher sensitivity. For alkalielectrophoresis, omit rinse in TBE and use cold Alkali Solution (pH 12.1). Adjust volume ofAlkali Solution so that the current is approximately 300 mA and perform electrophoresis at4° C or 16° C for 20 - 40 minutes. Temperature control during alkali electrophoresis ishighly recommended to minimize background and improve reproducibility.

10.Gently tap off excess solution and dip the slide in 70% ethanol for 5 minutes.

11.Air dry samples. Drying brings all the cells to a single plane which facilitates observation.At this stage samples may be stored at room temperature with desiccant prior to scoring.

12. Place 50 �L of diluted SYBR Green I (Reagent Preparation, Step 10) onto each circle ofdried agarose for 1 - 5 minutes.

Note: The CometAssay Silver Staining Kit (Catalog # TA4251) is also available. Silverstaining allows visualization of comets on any transmission light microscope andpermanently stains the samples for archiving and long term storage. It is recommendedthat samples be dried before silver staining.

13.View the slide by epifluorescence microscopy (SYBR Green I maximum excitation andemission are 494 nm/521 nm, respectively. A Fluorescein filter is adequate). If fadingoccurs, apply 50 �L of anti-fade solution (Reagent Preparation, step 11).

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CONTROLSControls should be included in every experiment. Two samples of untreated cells incubatedwith T4-Endo V and T4-Endo V FLARE Reaction Buffer 1 should always be processed tocontrol for endogenous levels of damage within cells and damage that may occur duringsample preparation. Each FLARE slide has three sample areas providing a convenient formatfor comparing samples with and without T4-Endo V treatment.

If you require a sample that will be positive for comet tails, treat cells with freshly prepared10 - 100 �M hydrogen peroxide or 5 - 25 �M KMnO4 for 20 minutes at 2 - 8° C. Hydrogenperoxide or KMnO4 treatment will generate significant oxidative damage/DNA adducts in themajority of cells. Note that the dimensions and characteristics of the comet tail, as aconsequence of hydrogen peroxide or KMnO4 treatment, may be different from those inducedby the damage under investigation.

DATA ANALYSISWhen excited (425 - 500 nm), the DNA-bound SYBR Green I emits green light. In healthy cells,the fluorescence is confined to the nucleoid: undamaged DNA is supercoiled and thus does notmigrate very far from the nucleoid under the influence of an electric current. In cells that haveaccrued damage to the DNA, the alkali treatment unwinds the DNA, releasing fragments thatmigrate from the cell when subjected to an electric field. The negatively charged DNA migratestoward the anode and the extrusion length reflects increasing relaxation of supercoiling, whichis indicative of damage. When using TBE as the electrophoresis buffer, the length of the comettail may be used to correlate with DNA damage. When using alkaline electrophoresisconditions, the distribution of DNA between the tail and the head of the comet is used toevaluate the degree of DNA damage. The characteristics of the comet tail including length,width, and DNA content may also be useful in assessing qualitative differences in the type ofDNA damage.

Qualitative AnalysisThe comet tail can be scored according to DNA content (intensity). The control (untreatedcells) should be used to determine the characteristics of data for a healthy cell. Scoringcan then be made according to nominal, medium, or high intensity tail DNA content. Atleast 75 cells should be scored per sample.

Quantitative AnalysisThere are several image analysis systems that are suitable for quantitation of CometAssaydata. The more sophisticated systems include a microscope, camera, and computeranalysis package. These systems can be set up to establish the length of DNA migration,image length, nuclear size, and calculate the tail moment. At least 75 randomly selectedcells should be analyzed per sample.

A list of commercial software packages is available from R&D Systems.

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TROUBLESHOOTING GUIDE

Problem Cause Action

Majority of cells in untreatedcontrol sample withoutT4-Endo V treatment have largecomet tails.

Unwanted damage to cellsoccurred in culture or in samplepreparations.

Intracellular endonucleaseactivity.

LMAgarose too hot.

Check morphology of cells toensure healthy appearance.

Handle cells or tissues gently toavoid physical damage.

Keep cells on ice and preparecell samples immediately beforecombination with moltenLMAgarose.

Cool LMAgarose to 37° Cbefore adding cells.

Majority of cells in untreatedcontrol sample withoutT4-Endo V treatment havesmall to medium comet tails.

Endogenous oxidative damageor endonuclease activity isdamaging DNA during samplepreparation.

Ensure lysis solution wasthoroughly chilled before use.

Add DMSO to any cell samplethat may contain heme groups.

Ensure PBS used is calciumand magnesium free.

Work under dimmed lightconditions or under yellow light.

No evidence of comet tail inpositive control (e.g. 100 �Mhydrogen peroxide for20 minutes on ice).

No damage to DNA.

Sample was not processedcorrectly.

Use freshly prepared reagentsto induce damage.

Ensure each step in protocolwas performed correctly. Failureto lyse, denature in alkali, or toproperly performelectrophoresis may generatepoor results.

Comet tails present but notsignificant in positive control.

Insufficient denaturation inAlkali Solution.

Insufficient electrophoresis time.

Buffer level duringelectrophoresis too high.

Increase time in Alkali Solutionup to 1 hour.

Increase time of electrophoresisup to 20 minutes. Maintainvoltage at 1 volt/cm.

Remove some buffer fromelectrophoresis chamber. If toomuch TBE buffer is present,migration will not occur.

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Problem Cause Action

Cells in LMAgarose did notremain attached to FLARESlide.

Cells were not washed toremove medium beforecombining with LMAgarose.

Agarose percentage was toolow.

LMAgarose not fully set beforesamples were processed.

LMAgarose unevenly set onslide.

The pH of the medium andcarry-over of serum proteinsetc. can reduce the adhesion ofthe agarose. Resuspend cells in1X PBS.

Do not increase the ratio of cellsto molten agarose by more than1:10 (e.g. add 50 �L of cellsuspension to 500 �L of moltenLMAgarose).

Ensure 0.5 mm dried ring due toagarose disc retraction is seenat edge of sample area. Extendgelling time to 30 minutes at2 - 8° C.

Spread the agarose with theside of a pipet tip to ensureuniformity of agarose disc andbetter adherence.

Majority of cells in untreatedsamples have large comet tailswith obliterated nuclei afterexposure to T4-Endo V.

The concentration of T4-Endo Vis too high.

Incubation time with the DNArepair enzyme is too long.

Endogenous DNA damage dueto handling conditions.

Titrate down the T4-Endo V toreduce or eliminate the comettails in the untreated cells.Altering the enzymeconcentration and incubationtimes are necessary tomaximize the difference incomet tail size between treatedand untreated cells.

Reduce incubation times.

Refer to first troubleshootingsection on previous page.

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WARNINGS� FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.� The physical, chemical, and toxicological properties of the products contained in the

FLARE Assay Kit may not have been fully investigated. Therefore, the use of gloves, labcoats, and eye protection while using any of these chemical reagents is recommended.R&D Systems assumes no liability for damage resulting from handling or contact withthese products.

� Lysis Solution contains 1% sodium lauryl sarcosinate which is an irritant. In case of eye orskin contact, wash thoroughly under running water. In case of ingestion, rinse mouth withwater and seek medical advice.

� SYBR Green I contains DMSO. Please refer to Material Safety Data sheets (MSDS).

APPENDICES

A. Reagents and Buffer Composition

10X TBE108 g Tris Base55 g Boric Acid9.3 g EDTA (disodium salt)Dissolve in 900 mL of deionized water.Adjust volume to 1 liter and autoclave.Store at room temperature.

25X FLARE Buffer 1250 mM HEPES-KOH, pH 7.42.5 M KCI250 mM EDTA

REC Dilution Buffer10 mM HEPES-KOH, pH 7.4100 mM KCI0.1 mg/mL BSA50% glycerol

100X BSA additiveProprietary stabilizer reagent

Lysis Solution2.5 M sodium chloride100 mM EDTA, pH 1010 mM Tris base1% sodium lauryl sarcosinate1% Triton X-100

Comet LMAgarose1% low melting point agarose1X PBS

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B. Suggestions for Assay Optimization

Under optimal conditions for T4-Endo V FLARE, there will be an increase in the comet tailmoment of cells containing thymine dimers incubated with T4-Endo V, compared to cellswithout T4-Endo V treatment. Control cells that have been processed through the assay buthave the T4-Endo V treatment omitted, may still generate comets due to the presence of othertypes of damage detected directly by the comet assay, e.g. endogenous abasic sites. If thecomet tails in the control cells are too large it can be difficult to detect small changes as aconsequence of T4-Endo V treatment. Parameters to consider for optimization include:

A) TemperatureDuring the process of DNA repair, abasic sites, nicks, and gaps in the DNA can beintroduced that may generate comets in the FLARE assay. Repair is inhibited by coldtemperatures; therefore, it is important to keep the cells and solutions cold (2 - 8° C or onice) until cell lysis and deproteination are complete. Some repair has been reported evenat 2 - 8° C; therefore, cells should not be held at 2 - 8° C for long periods of time beforesample preparation. FLARE may be used to monitor the repair process in which case thecells would be incubated at physiological temperatures to allow repair, before analysis byFLARE.

B) Enzyme selection and titrationT4-Endo V recognizes cis-syn thymine dimers and has an associated AP-lyase activity.Other FLARE Kits are available that may be more appropriate for the type of damageunder investigation. We recommend that you titrate T4-Endo V to optimize the differencesin tail moment between untreated and T4-Endo V-treated cells. Typical final dilutions ofT4-Endo V used are 1:100, 1:500, and 1:1000. If necessary, up to 10 �L of undilutedenzyme can be applied in 100 �L of T4-Endo V FLARE Reaction Buffer (i.e. A final dilutionof 1:10).

C) Incubation times with T4-Endo VOptimum temperature for T4-Endo V is 37° C. Vary the incubation time at 37° C up to1 hour with T4-Endo V to optimize the differences in tail moment between untreated andT4-Endo V-treated cells.

D) Health of your cellsYour cells must be at least 95% viable as measured by Trypan Blue exclusion. The cometassay should not be used for detection of DNA damage under conditions of highcytotoxicity when there is massive secondary DNA damage. Adherent cells should begently trypsinized prior to analysis. Note that extensive trypsinization may inducenon-specific DNA damage and repair and, therefore, high background.

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REFERENCES1. Nia, A.B. et al. (2001), Carcinogenesis 22:395.

2. Lemay, M. et al. (1999), BioTechniques 27(4):846.

3. Angelis, K.J. et al. (1999), Electrophoresis 20:2133.

4. Morris, E.J. et al. (1999), BioTechniques 26:282.

5. Malyapa, R.S. et al. (1998), Radiation Res. 149:396.

6. Henderson, L. et al. (1998), Mutagenesis 13:89-94.

7. Visvardis, E.E. et al. (1997), Mutation Res. 383:71.

8. Fairbairn, D.W. et al. (1995), Mutation Res. 339:37.

9. Collins, A.R. et al. (1995), Mutation Res. 336:69.

10. Singh, N.P. et al. (1988), Exp. Cell Res. 175:184.

11. Östling, O. and K.J. Johanson, (1984), Biochem. Biophys. Res. Commun. 123:291.

SYBR Green I Nucleic acid gel stain is a registered trademark of Molecular Probes, Inc. and is sold under license from

Molecular Probes, Inc. under US patent numbers 5436134 and 5658751. For use in a comet assay for internal research

and development only, where research and development use expressly excludes the use of this product for providing

medical, diagnostic or any other testing analysis or screening services or providing clinical information or clinical analysis, in

return for compensation on a per-test basis, and research and development use expressly excludes incorporation of this

product into another product for commercialization even if such other product would be commercialized for research and/or

development use.

CometAssay, FLARE and REC are trademarks of Trevigen, Inc.

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NOTES

12.02 750132.1 4/04

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