OxiSelect™ Comet Assay Kit (3-Well Slides)

1. OxiSelect™ 3-Well Comet Slides : Five slides.
2. OxiSelect™ Comet Agarose : One sterile 15 mL bottle.
3. Vista Green DNA Dye, 10000X : One 5 μL vial.
4. EDTA Solution, 500 mM : One 50 mL bottle.
5. 10X Lysis Solution : One 20 mL bottle.

1. NaCl powder
2. NaOH pellets
3. 10 N NaOH for pH adjustment
4. DMSO (optional)
5. 70 % Ethanol
6. TE Buffer (10 mM Tris, pH 7.5, 1 mM EDTA)
7. PBS (without Mg2+ and Ca2+)
8. EDTA (disodium salt)
9. DI H2O
10. 37 °C and boiling water baths
11. Horizontal electrophoresis chamber
12. Adjustable single channel micropipettes with disposable tips
13. Epifluorescence microscope with FITC filter 4

• Useful screening tool for various types of DNA damage
• Slides are specially treated for adhesion of low-melting agarose
• Easy visualization by epifluorescence microscopy

• OxiSelect™ Comet Agarose: Heat the Comet Agarose bottle at 90-95 °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 H2O. 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 1.2 g 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. Alkaline 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 5 Boric Acid 5.5 g 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 >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. 2. 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. 3. Prepare cell samples, including controls, as follows:
• Suspension Cells: Centrifuge cells at 700 x g for 2 minutes and discard supernatant. Wash cell pellet once with ice-cold PBS (without Mg2+ and Ca2+), centrifuge, and discard the supernatant. Finally, resuspend the cells at 1 x 105 cells/mL in ice-cold PBS (without Mg2+ and Ca2+).
• 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 Mg2+ and Ca2+), centrifuge, and discard the supernatant. Finally, resuspend the cells at 1 x 105 cells/mL in ice-cold PBS (without Mg2+ and Ca2+).
• Tissue Preparation: Using dissection scissors, mince a small piece of tissue in 1-2 mL of ice cold PBS containing 20 mM EDTA (without Mg2+ and Ca2+). 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 105 cells/mL in ice-cold PBS (without Mg2+ and Ca2+). 4. Combine cell samples with Comet Agarose (step 2) at 1:10 ratio (v/v), titrate to mix, and immediately pipette 75 μL/well onto the OxiSelect™ Comet Slide. Ensure complete well coverage by spreading the suspension over the well with the pipette tip. Note: For multiple samples, maintain suspensions at 37 °C to avoid gelation. Titrate samples again just prior to slide addition. 5. Maintaining the slide horizontally, transfer the slide to 4 °C in the dark for 15 minutes. 6 6. Carefully, transfer the slide to a small basin/container containing pre-chilled Lysis Buffer (~25 mL/slide). Immerse the slide in the buffer for 30-60 minutes at 4 °C in the dark. 7. Carefully, aspirate the Lysis Buffer from the container and replace with pre-chilled Alkaline Solution (~25 mL/slide). Immerse the slide in the solution for 30 minutes at 4 °C in the dark.

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.
2. 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.
3. 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)
4. Maintaining the slide horizontally, carefully transfer the slide from the electrophoresis chamber to a clean, small basin/container containing pre-chilled DI H2O (~25 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 for 5 minutes.
6. Maintaining the slide horizontally, remove the slide from the 70 % Ethanol and allow to air dry.
7. Once the agarose and slide is completely dry, add 100 μL/well of diluted Vista Green DNA Dye (see Preparation of Reagents). Incubate at room temperature for 15 minutes.
8. 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.
2. 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.
3. Maintaining the slide horizontally, carefully transfer the slide from the electrophoresis chamber to a clean, small basin/container containing pre-chilled DI H2O (~25 mL/slide). Immerse the slide for 2 minutes, aspirate, and then repeat twice more.
4. 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.
6. Once the agarose and slide is completely dry, add 100 μL/well of diluted Vista Green DNA Dye (see Preparation of Reagents). Incubate at room temperature for 15 minutes.
7. View slides by epifluorescence microscopy using a FITC filter. 7

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 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 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) Figure 3: Typical Damaged DNA in Comet Assay. 8