CometAssay™

Reagent Kit for Single Gel

Catalog Number: TA800

50 Tests (2 tests/slide)

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 CONTENTS Contents Page INTRODUCTION 2 REAGENTS PROVIDED ...... 3 MATERIALS REQUIRED BUT NOT PROVIDED 3 REAGENT PREPARATION...... 4 SAMPLE PREPARATION AND STORAGE 6 ASSAY PROTOCOL ...... 7 COMETASSAY PROTOCOL 8 TBE Electrophoresis ...... 9 Alkali Electrophoresis 9 Silver of CometSlides ...... 10 DATA ANALYSIS 11 TROUBLESHOOTING GUIDE...... 12 APPENDICES 13 Neutral CometAssay ...... 13 Storing CometSlides 14 Reagent and Buffer Composition ...... 14 REFERENCES 14 WARNINGS ...... 15

MANUFACTURED FOR AND DISTRIBUTED BY: R&D Systems, Inc. TELEPHONE: (800) 343-7475 614 McKinley Place NE (612) 379-2956 Minneapolis, MN 55413 FAX: (612) 656-4400 United States of America E-MAIL: [email protected]

R&D Systems Europe, Ltd. 19 Barton Lane TELEPHONE: +44 (0)1235 529449 Abingdon Science Park FAX: +44 (0)1235 533420 Abingdon, OX14 3NB E-MAIL: [email protected] United Kingdom

R&D Systems GmbH Borsigstrasse 7 TELEPHONE: +49 (0)6122 90980 65205 Wiesbaden-Nordenstadt FAX: +49 (0)6122 909819 Germany E-MAIL: [email protected]

R&D Systems Europe 77 boulevard Vauban FREEPHONE: +0800 90 72 49 59800 LILLE FAX: +0800 77 16 68 France E-MAIL: [email protected] INTRODUCTION The CometAssay™, or single cell gel electrophoresis assay, provides a simple and effective method for evaluating DNA damage to cells. The principle of the assay is based upon the ability of denatured cleaved DNA fragments to migrate out of the cell under the influence of an electric potential, whereas undamaged supercoiled DNA remains within the confines of the when a current is applied. Evaluation of the DNA “” tail shape and migration pattern allows for assessment of DNA damage. The method involves the immobilization of cells in a bed of low melting point , followed by gentle cell . After treatment with alkali to denature the DNA and hydrolyze sites of damage, electrophoresis, and staining of the sample with a fluorescent DNA intercalating dye, the sample is visualized under the by epifluorescence. As an alternative for researchers who do not have access to an epifluorescence microscope, silver staining allows the use of a standard transmission microscope for data analysis. The CometAssay is optimized for use with TBE buffer for electrophoresis. TBE circumvents the problems associated with use of non-buffered alkali for electrophoresis, such as poor DNA migration due to saturation of charge by sodium ions, and difficulties in controlling voltage. TBE allows for electrophoresis times of only 10 minutes and also allows tail length, rather then the tail moment, to be used to quantitate data. The use of tail length for quantitation avoids the need to normalize tail moment with total DNA. Qualitative data may be generated if the are scored according to categories of small to large. Quantitative and statistical data can readily be generated by analysis of the results using one of several commercially available image analysis software packages that can calculate tail length and tail moment. The CometAssay’s CometSlide™ is specially treated to promote adherence of low melting point agarose. This eliminates the time consuming and unreliable traditional method of preparing base layers of agarose. The use of CometSlides shortens assay time, and allows the Ò rapid and reliable analysis of large numbers of samples. SYBR Green I provides improved sensitivity compared to ethidium bromide for DNA visualization.

2 REAGENTS PROVIDED

Component Number Component Amount Provided Storage Temperature 4250-050-01 Lysis Solution 2 x 500 mL

4250-050-02 Comet LMAgarose (LMA) 15 mL Store at room 4250-050-03 CometSlide 25 each temperature 4250-050-04 200 mM EDTA, pH 10 12.5 mL SYBRÒ Green I 4250-050-05 5 mL £ -20° C nucleic acid gel stain

MATERIALS REQUIRED BUT NOT PROVIDED Reagents ++ ++ · 10X phosphate-buffered saline (PBS), Ca and Mg free · NaOH Pellets · Dimethylsulfoxide (optional) · 10X TBE Buffer (required for neutral electrophoresis) · 0.5 M EDTA, pH 8.0 (required for alkaline electrophoresis) · Silver staining reagents (optional - refer to page 10 for details) · TE Buffer (10 mM Tris pH 7.5, 1 mM EDTA) · Methanol, Ethanol (optional) · High quality deionized water (dH2O) Equipment · 1 - 20 mL, 20 - 200 mL, 200 - 1000 mL pipettes · 1 Liter graduated cylinder · Boiling water bath and 37° C water bath · Horizontal electrophoresis apparatus · Epifluorescence microscope equipped with FITC filter · 2 - 8° C refrigerator · Peristalic pump Disposables · Pipette tips · Serological pipettes

3 REAGENT PREPARATION Reagents marked with an asterisk (*) should be prepared just before use. Wear gloves, lab coat and eye protection when handling any chemical reagents. 1. 1X PBS, Ca++ and Mg++ free Dilute 10X PBS with deionized water to prepare 1X PBS. Store at room temperature. 2. Lysis Solution For up to 10 slides (2 samples per slide) prepare:

Lysis Solution (Component # 4250-050-01) 40 mL DMSO 4 mL (optional)

Chill at 2 - 8° C, or on ice, for at least 20 minutes before use. Note: The addition of DMSO is optional and is required only for samples containing heme, such as blood cells or tissue samples. Lysis Solution contains 2.5 M NaCl, 100 mM EDTA, 10 mM Tris Base, 1% sodium lauryl sarcosinate, and 1% Triton X-100. 3. Comet LMAgarose The Comet LMAgarose is ready to use once molten. Loosen the cap to allow for expansion then heat the bottle in a 90 - 100° C water bath for 5 minutes, or until the agarose is molten (Caution: Microwaving is not recommended). Place the bottle in a 37° C water bath for at least 20 minutes to cool. The Comet LMAgarose will remain molten at 37° C. Comet LMAgarose contains 1% low melting point agarose in 1X PBS. 4. Alkaline Solution, pH > 13* Wear gloves when preparing and handling the Alkaline Solution. For 50 mL of Alkaline Solution, combine:

NaOH Pellets 0.6 g 200 mM EDTA, pH10 250 mL Deionized water 49.75 mL

Stir until fully dissolved. The solution will warm during preparation - allow to cool to room temperature before use.

4 5. Electrophoresis Solution Prepare one of the following electrophoresis solutions based on the sensitivity of assay desired. A. 1X TBE Buffer 1X TBE is used for electrophoresis because of its buffering capacity. To prepare 10X TBE, dissolve:

Tris base 108 g Boric acid 55 g EDTA (disodium ) 9.3 g Deionized water 900 mL

Adjust volume to 1 liter and autoclave. Store at room temperature. Dilute the 10X TBE to 1X in deionized water to prepare working strength buffer. Or B. Alkaline Electrophoresis Solution pH > 13 (300 mM NaOH, 1 mM EDTA)* Wear gloves, lab coat and eye protection when preparing and handling the Alkaline Solution. Prepare a stock solution of 500 mM EDTA, pH 8.0. For 1 liter of electrophoresis solution add:

NaOH pellets 12 g 500 mM EDTA, pH 8.0 2 mL Deionized water to 1 liter (after NaOH is dissolved)

Adjust the volume prepared based on the dimensions of your electrophoresis apparatus. Use of freshly made solution is recommended. 6. SYBR Green I Staining Solution Prepare SYBR Green I Staining Solution from the SYBR Green I concentrate provided.

SYBR Green I (Component # 4250-050-05) 1 mL TE Buffer, pH 7.5 (TE: 10 mM Tris pH 7.5, 1mM EDTA) 10 mL

The diluted stock is stable for several weeks when stored at 2 - 8° C in the dark. 7. Anti-fade Solution Prepare if fading of samples occurs. In a 50 mL tube, mix until dissolved:

P-Phenylenediamine dihydrochloride 500 mg 1X PBS 4.5 mL

Add approximately 400 mL of 10 N NaOH dropwise while stirring to a pH level of 7.5 - 8.0. Add 1X PBS to increase the volume to 5 mL and add 45 mL of glycerol for a final volume of 50 mL. Vortex mixture thoroughly and apply 10 mL per sample. Cover samples with coverslips. Nail polish may be used to seal coverslip. Re-staining of slides is not recommended. Store Anti-fade solution at -20° C for up to one month. Darkening of solution may occur.

5 SAMPLE PREPARATION AND STORAGE Cell samples should be prepared immediately before starting the assay, although success has been obtained using cryopreserved cells (see page 7). Cell samples should be handled under dimmed or yellow light to prevent DNA damage from light. Buffers should be chilled to 2 - 8° C or placed on ice to inhibit endogenous damage from occurring during sample preparation and to inhibit repair in the unfixed cells. PBS must be calcium and magnesium free to inhibit endonuclease activities. The appropriate controls should also be included (refer to the Controls section). Optimal results in the CometAssay are usually obtained with 500 - 1000 cells per CometSlide sample area. Using 50 mL of a cell suspension at 1 x 105 cells/mL combined with 500 mL of LMAgarose will provide the correct agarose concentration and cell density for optimal results when plating 75 mL per sample. Suspension Cells Cell suspensions are harvested by centrifugation. Cells may be resuspended at 1 x 105 cells/mL in either ice cold 1X PBS (Ca++ and Mg++ free) or in ice cold medium. If the cell concentration is high enough prior to harvesting, the cells may be added directly into the molten LMAgarose. Although applying cells in the more common media used for onto CometSlides has not affected performance, some media may contain additives that will adversely affect results. Adherent Cells Scrape cells using a rubber policeman. Transfer cells and medium to a centrifuge tube, perform cell count, then pellet cells. Wash once in ice cold medium or in ice cold 1X PBS (Ca++ and Mg++ free). Resuspend at 1 x 105 cells/mL in ice cold medium or in ice cold 1X PBS (Ca++ and Mg++ free). Tissue Preparation Place a small piece of tissue into 1 - 2 mL of ice cold 1X PBS (Ca++ and Mg++ free), 20 mM EDTA. Using small dissecting 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 by centrifugation, and resuspend at 1 x 105 cells/mL in ice cold 1X PBS (Ca++ and Mg++ free).For blood rich organs (e.g., liver, spleen), chop tissue into large pieces (1 - 2 mm3), let settle for 5 minutes, then aspirate and discard medium. Add 1 - 2 mL of ice cold 20 mM EDTA in 1X PBS (Ca++ and Mg++ free), mince the tissue into very small pieces and let stand for 5 minutes. Recover the cell suspension, avoiding transfer of debris. Count cells, pellet, and resuspend at 1 x 105 cells/mL in ice cold 1X PBS (Ca++ and Mg++ free).

Controls Controls should be included in each experiment. A sample of untreated cells should always be processed to control for endogenous levels of damage within cells, and for damage that may occur during sample preparation. Control cells and treated cells should be handled in an identical manner. If UV damage is being studied, the cells should be kept in low level yellow light during processing. If you require a sample that will be positive for comet tails, treat cells with 100 mM or 25 mM KMnO4 for 10 minutes at 2 - 8° C. Treatment will generate significant oxidative damage in the majority of cells, thereby providing a positive control for each step in the CometAssay. Note that the dimensions and characteristics of the comet tail, as a consequence of hydrogen peroxide treatment, may be different to those induced by the damage under investigation.

6 Method for Cryopreservation of Cells Prior to CometAssay Certain cells, e.g. lymphocytes, may be successfully cryopreserved prior to performing CometAssay [Visvardis, E.E. et al. (1997) Mutation Res. 383:7180]. A pilot study should be performed to determine if cryopreservation 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 has melted. 6. Transfer to 15 mL of prechilled 40% (v/v) medium, 10% (w/v) dextrose, 50% (v/v) fetal calf serum. 7. Centrifuge at 200 x g for 10 minutes at 2 - 8° C. 8. Resuspend in ice cold 1X PBS (Ca++ and Mg++ free) and proceed with CometAssay. ASSAY PROTOCOL Both protocols provided are for alkaline unwinding conditions. The electrophoresis conditions will determine the sensitivity of the assay. TBE electrophoresis or Neutral electrophoresis after alkaline unwinding will detect single-stranded DNA breaks, double-stranded DNA breaks, and may detect a few apurinic sites and apyrimidinic sites. Alkaline electrophoresis will detect single-stranded DNA breaks, double-stranded DNA breaks, and the majority of apurinic sites, apyrimidinic sites as well as alkaline labile DNA adducts (e.g. phosphoglycols, phosphotriesters). The CometAssay has been reported to detect DNA damage associated with low doses (0.6 cGy) of gamma irradiation, providing a simple technique for quantitation of low levels of DNA damage. Prior to performing the CometAssay, a viability assay should be performed to determine the dose of the test substance that gives at least 75% viability. False positives may occur when high doses of cytotoxic agents are used. For information on performing the Neutral CometAssay that will predominantly detect double-stranded DNA breaks, refer to the Appendix on page 13. For cryopreservation of cells, fixing the CometSlide samples, and storage, refer to the Sample Preparation and Storage section, pages 6 - 7. The CometAssay requires approximately two hours to complete, including the incubations and electrophoresis. Once the cells or tissues have been prepared, the procedure is not labor intensive. The Lysis Solution may be chilled and the LMAgarose melted while the cell and tissue samples are being prepared.

7 COMETASSAY PROTOCOL All steps are performed at room temperature unless otherwise specified. Work under dimmed or yellow light to prevent damage from UV light. 1. Prepare Lysis Solution (refer to the Reagent Preparation section, page 4) and chill at 2 - 8° C or on ice for at least 20 minutes before use. 2. Melt LMAgarose in boiling water for 5 minutes, with the cap loosened. Place bottle in a 37° C water bath for at least 20 minutes to cool. The temperature of the agarose is critical or the cells may undergo heat shock. Heat blocks are not recommended for regulating the temperature of the agarose. 3. Combine cells at 1 x 105/mL with molten LMAgarose (at 37° C) at a ratio of 1:10 (v/v) and immediately pipette 75 mL onto a CometSlide. Use the side of the pipette tip to spread the agarose/cells over sample area.

Comet LMAgarose (molten and at 37° C from step 2) 500 mL Cells in 1X PBS (Ca++ and Mg++ free) or medium at 1 x 105 cells/mL 50 mL Note: If sample is not spreading evenly on the slide, warm the slide at 37° C before application. 4. Place slides flat at 2 - 8° C in the dark (e.g. place in refrigerator) for 10 minutes. A 0.5 mm clear ring appears at edge of CometSlide area. Increasing gelling time to 30 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 for 30 to 60 minutes. 6. Tap off excess buffer from the slide and immerse in freshly prepared Alkaline Solution, pH > 13 (refer to Reagent Preparation section, page 4). Wear gloves when preparing or handling this solution. 7. Leave CometSlide in Alkaline Solution for 20 to 60 minutes at room temperature in the dark. To perform TBE Electrophoresis, go to step 8. For Alkaline Electrophoresis, go to step 13.

8 For TBE Electrophoresis 8. Remove slide from Alkaline Solution, gently tap excess buffer from slide and wash by immersing in 1X TBE buffer for 5 minutes, 2 times (Reagent Preparation, page 4). 9. Transfer slide from 1X TBE buffer to a horizontal electrophoresis apparatus. Place slides onto a gel tray and align equidistant from the electrodes. Pour 1X TBE buffer until level just covers samples. Set power supply to 1 volt per cm (measured electrode to electrode). Apply voltage for 10 minutes. 10. Gently tap off excess TBE, and dip slide in 70% ethanol for 5 minutes. 11. Air dry samples. Drying brings all the cells into a single plane to facilitate observation. Samples may be stored at this stage. Store at room temperature with desiccant. Note: Samples may be fixed, dried and stored prior to scoring at this stage (refer to appendix). For viewing with a transmission light microscope, CometSlides may be silver stained (Silver Staining of CometSlides, page 10). 12. Proceed to Step 16. For Alkaline Electrophoresis 13. Transfer slide from Alkaline Solution to a horizontal electrophoresis apparatus. Place slides flat onto a gel tray and align equidistant from the electrodes. Carefully pour the Alkaline Solution until level just covers samples. Set the voltage to about 1 volt/cm. Add or remove buffer until the current is approximately 300 mA and perform electrophoresis for 20 - 40 minutes. Tips: Since the Alkaline Electrophoresis Solution is a non-buffered system, temperature control is highly recommended. Testing has shown great temperature fluctuations when conducting the alkaline electrophoresis at ambient temperature. To improve temperature control, the use of a large electrophoresis apparatus (25 - 30 cm between electrodes) is recommended along with recirculation of the electrophoresis solution. Alternatively, performing the electrophoresis at cooler temperatures (e.g. 16° C or 4° C) will diminish background damage, increase sample adherence at high and significantly improve reproducibility. Choose the method that is most convenient for your laboratory and always use the same conditions, power supplies and electrophoresis chambers for comparative analysis. 14. Gently tap off excess electrophoresis solution, rinse by dipping several times in dH2O, then immerse slide in 70% ethanol for 5 minutes. 15. Air dry samples. Drying brings all the cells into a single plane to facilitate observation. Samples may be stored at room temperature, with desiccant prior to scoring at this stage. Note: Samples may be fixed, dried and stored prior to scoring at this stage (refer to appendix). For viewing with a transmission light microscope, CometSlides may be silver stained (Silver Staining of CometSlides, page 10).

16. Place 50 mL of diluted SYBR Green I ( refer to Reagent Preparation, page 4) onto each circle of dried agarose. 17. View slide by epifluorescence microscopy (SYBR Green I’s maximum excitation and emission are 494 nm and 521 nm, respectively. Fluorescein filter is adequate).

9 Silver Staining of CometSlides Silver staining offers the opportunity to visualize comets on any transmission light microscope and allows for long term storage. 1. After electrophoresis or SYBR Green I staining, wash CometSlide twice in deionized water for 2 minutes each wash. 2. Dry the CometSlide at room temperature for 1 hour. 3. Fix slide for 10 minutes in 15% w/v trichloroacetic acid, 5% w/v zinc sulphate, 5% v/v glycerol. 4. Wash slide twice in deionized water for 2 minutes each wash. 5. Redry the CometSlide at room temperature for 1 hour or overnight. 6. Prepare 100 mL of staining solution just prior to use: · to 62 mL of a stirring solution A (5% w/v sodium carbonate) · add dropwise 32 mL of solution B (0.02% w/v silver nitrate, 0.02% w/v ammonium nitrate, 0.1% w/v tungstosilicid acid, 0.05% v/v formaldehyde, 5% w/v sodium carbonate). 7. Immerse the CometSlide for 10 minutes in 50 mL freshly prepared staining solution. 8. Immerse slide for additional 10 minutes in remaining staining solution or until a grey/brown color develops on the slide. 9. Wash slide twice in deionized water for 2 minutes each wash. 10. Stop reaction by immersing slide in 1% acetic acid solution for 5 minutes. 11. Wash slide twice in deionized water for 2 minutes each wash. 12. The silver stained slides may be air dried and stored at room temperature with desiccant. Note: Successful labeling can be achieved with commercially available silver staining kits, following the manufacturers instructions for staining dried agarose gels for DNA.

10 DATA ANALYSIS When excited by light (425 - 500 nm) the SYBR Green I that has bound to single and double stranded DNA emits green light. In healthy cells, the fluorescence is confined to the nucleus: undamaged DNA is supercoiled and thus does not migrate out of the nucleus under the influence of an electric current. In cells that have accrued damage to the DNA, the alkaline treatment unwinds the DNA and releases fragments that migrate from the cell when subjected to an electric field. The negatively charged DNA migrates toward the anode and the extrusion length reflects increasing relaxation of supercoiling, indicative of damage. The length of the comet tail correlates with the degree of DNA damage. When using TBE as the electrophoresis buffer, the length of the comet tail may be used to correlate with DNA damage. When using alkali electrophoresis conditions, the distribution of DNA between the tail and the head of the comet should be used to evaluate 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 of DNA damage. Qualitative analysis The comet tail can be scored according to length. The control (untreated cells) should be used to determine the characteristics of data for a healthy cell. Scoring can then be made according to nominal, medium or long tail. At least 100 cells should be scored per sample. Quantitative analysis There are several image analysis systems that are suitable for quantitation of Comet assay data. The more sophisticated systems include the microscope, camera and computer analysis package. The system can be set up to establish the length of DNA migration, the image length, the nuclear size, and will calculate the tail moment. At least 75 randomly selected cells should be analyzed per sample. A list of commercial software packages is available from R&D Systems.

11 TROUBLESHOOTING GUIDE

Problem Cause Action Majority of cells in untreated Unwanted damage to cells occurred Check morphology of cells to ensure control sample have large in culture or in sample preparations. healthy appearance. comet tails. Handle cells or tissues gently to avoid physical damage. · Electrophoresis solution too hot Control tempertaure by recirculating the electrophoresis solution or performing the assay at less than 20° C. · Intracellular activity Keep cells on ice and prepare cell samples immediately before combination with molten LMAgarose. · LMAgarose too hot Cool LMAgarose to 37° C before adding cells. Do not use a heat block. Majority of cells in untreated Endogenous oxidative damage or Ensure lysis solution was thoroughly control sample have small to endonuclease activity after sample chilled before use. medium comet tails. preparation is damaging DNA. Add DMSO to any cell sample that may contain heme groups. Ensure PBS used is calcium and magnesium free. Work under dimmed or yellow light conditions. In positive control (e.g. 100 mM hydrogen peroxide No damage to DNA. Use fresh hydrogen peroxide to for 30 minutes on ice) no induce damage. evidence of comet tail.

Sample was not processed correctly. Ensure each step in protocol was performed correctly. Failure to lyse, denature in alkaline (optional), or to properly perform electrophoresis, may generate poor results. Comet tails present but not Insufficient denaturation in Alkali Increase time in Alkaline Solution up significant in positive control. Solution. to one hour. Insufficient electrophoresis time. Increase time of electrophoresis up to 20 minutes for TBE and up to 1 hour for alkaline electrophoresis. Cells in LMAgarose did not Electrophoresis solution too hot Control tempertaure by recirculating remain attached to the electrophoresis solution or CometSlide. performing the assay at less than 20° C. Cells were not washed to remove The pH of medium and carry over of medium before combining with serum etc. can reduce the LMAgarose. adhesion of the agarose. Resuspend cells in 1X PBS. Agarose percentage was too low. Do not increase ratio of cells to molten agarose by more than 1:10 e.g. add 50 mL of cell suspension to 500 mL of molten LMAgarose. LMAgarose not fully set before Ensure 0.5 mm dried ring due to samples processed. agarose disc retraction is seen at edge of CometSlide area. LMAgarose unevenly set on slide. Spread the agarose with the side of a pipette tip to ensure uniformity of agarose disc and better adherence.

12 APPENDICES Neutral CometAssay The CometAssay may be performed using neutral conditions. Without treatment with Alkaline Buffer, the Neutral CometAssay will detect mainly double stranded breaks and can be useful for assessing the DNA fragmentation associated with apoptosis. 1. Prepare Lysis Solution (see Reagent Preparation page 4) and chill at 2 - 8° C or on ice for at least 20 minutes before use. 2. Melt LMAgarose in boiling water for 5 minutes, with the cap loosened, then cool in a 37° C water bath for at least 20 minutes. 3. Combine cells at 1 x 105/mL with molten LMAgarose (at 37° C) at a ratio of 1:10 (v/v) and immediately pipette 75 mL onto CometSlide. Use side of pipette tip to spread agarose/cells over sample area.

Comet LMAgarose, molten and at 37° C (step 2) 500 mL Cells in 1X PBS (Ca++ and Mg++ free) or medium at 1 x 105/mL 50 mL

Note: If sample is not spreading evenly on the slide, warm the slide at 37° C before application. The sample volume can also be increased to 75 mL per area. 4. Place slides flat at 2 - 8° C in the dark (e.g. place in refrigerator) for 10 minutes. A 0.5 mm clear ring appears at inside edge of CometSlide area. Increasing gelling time to 30 minutes improves adherence of samples in high humidity environments. 5. Immerse slide in prechilled (step 1) Lysis Solution and leave on ice or at 2 - 8° C for 30 minutes. 6. Remove slide from Lysis Buffer, tap excess buffer from slide and wash slide by immersing in 50 mL of 1X TBE buffer (refer to Reagent Preparation section, page 4). 7. Transfer slides from 1X TBE buffer, and place flat onto a gel tray submerged in 1X TBE buffer in a horizontal electrophoresis apparatus. Align slides equidistant from the electrodes. Set power supply to 1 volt per cm (measured electrode to electrode). Apply voltage for 10 - 20 minutes. 8. Tap off excess TBE, rinse slide briefly in deionized water. 9. Dip slide in 70% ethanol for 5 minutes. 10. Air dry samples. Drying brings all the cells into a single plane to facilitate observation. Samples may be stored at room temperature, with desiccant prior to scoring at this stage. Note: Samples may be fixed, dried and stored prior to scoring at this stage (refer to page 14). For viewing with a transmission light microscope, CometSlides may be silver stained (see Silver Staining of CometSlides, page 10). 11. Place 50 mL of diluted SYBR Green I (refer to Reagent Preparation, page 4) onto each circle of dried agarose. 17. View slide by epifluorescence microscopy (SYBR Green I maximum excitation and emission are 494 nm and 521 nm, respectively Fluorescein filter is adequate).

13 Storing CometSlides Before Scoring Comets CometSlides may be stored prior to scoring by fixing the samples in methanol and drying after an ethanol wash. The slides may be stored for several months in a low humidity atmosphere prior to staining with SYBR Green I. 1. After electrophoresis, immerse CometSlide in ice cold 100% methanol for 5 minutes. 2. Immerse in 100% ethanol for 5 minutes. 3. Drain excess alcohol and lay CometSlide flat to dry at room temperature. 4. Store at room temperature with desiccant. 5. For scoring, cover sample areas with diluted SYBR Green I and proceed with viewing. Reagent and Buffer Composition Lysis Solution (4250-050-01) 2.5 M sodium chloride 100 mM EDTA, pH 10 10 mM Tris base 1% sodium lauryl sarcosinate 1.0% Triton X-100

LMAgarose (4250-050-02) 1.0% low melting point agarose in 1X PBS

SYBR Green I (4250-050-05) 10,000X concentrate in DMSO REFERENCES 1. Fairbairn, D.W. et al. (1995) Mutation Res. 339:37. 2. Lemay, M. and K.A. Wood, (1999) BioTechniques 27:846. 3. Angelis, K.J. et al. (1999) Electrophresis 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.A. et al. (1998) Mutagenesis 13:89. 7. Collins, A.R. et al. (1995) Mutation Res. 336:69. 8. Singh, N.P. et al. (1988) Cell Res. 175:184. 9. Ostling, O and K.J. Johanson, (1984) Biochem. Biophys Res Commun. 123:291.

14 WARNINGS

HAZARDOUS INGREDIENTS Lysis Solution contains 1% sodium lauryl sarcosinate which is an irritant. In case of eye or skin contact, wash throughly under running water. In case of ingestion, rinse mouth with water and seek medical attention. SYBR Green I contains DMSO. Please consult the MSDS sheets for details.

HANDLING PRECAUTIONS The physical, chemical and toxicological properties of the products contained within the CometAssay Kit may not have been fuly investigated. Therefore, the use of gloves, lab coats and eye protection while using any of these chemical reagents is recommended.

EMERGENCY EXPOSURE PROCEDURES In case of exposure to reagent solutions, we recommend following these emergency first-aid procedures: Skin or eye contact: Wash with water for at least 15 minutes. Remove any contaminated clothing. Inhalation: Remove individual to fresh air. If breathing is difficult, give oxygen and call a physician. Ingestion: Rinse mouth with copious amounts of water and call a physician.

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.

Ò Tween 20 is a registered trademark of ICI Americas, Inc. CometAssay and CometSlide are trademarks of Trevigen, Inc.

07.99 750437.3 4/04

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