Comet Assay for the Detection of DNA Breaks Protocol

Introduction of SCGE for the Detection of DNA Breaks

The comet assay, also called the single-cell gel electrophoresis, is an invaluable tool for quantifying DNA damage in individual cells. The concept of DNA migrating in an agarose gel by an electric current was first introduced in 1984 by Ostling and Johanson as a method to measure single stranded breaks that caused a relaxation of the DNA supercoiled structure under neutral conditions. They also introduced the terms "comet" and "tail" as they described the shape of the displaced DNA and the tail moment (tail length and percentage of fluorescence) as an indicator of DNA migration. A modified version to quantify low levels of DNA damage in single cells after exposure to X-rays and hydrogen peroxide under alkaline conditions was published by Singh et al. in 1988. The assay relies on the principle that low-molecular-weight DNA fragments migrate away from damaged supercoiled DNA under an electric current, creating in a visible DNA fragment tail off of the intact nuclear DNA head. Individual comets can then be scored qualitatively using manual analysis or quantitatively by using software that determines the relative fraction of DNA in the comet tail. The comet assay protocol is relatively simple and can be completed in 1 day with as few as 1000 cells, making it useful for monitoring the efficacy of genotoxic agents, such as chemotherapeutics and radiation, and for determining the rate of DNA repair in cancer cells. Modifications to the comet assay have enabled the detection of DNA damage associated with specific cellular processes and genes. For instance, the use of bromodeoxyuridine in combination with the comet assay facilitates the detection of DNA breaks in replicating cells, and by employing lesion-specific enzymes sensitivity and specificity of the comet assay can be increased. In addition, in situ hybridization can be also combined with the comet assay to identify DNA breaks in individual genetic regions.

Despite its utility, there are several limitations the comet assay, which need to be discussed. Small variations in agarose concentration, voltage gradients, current, and electrophoresis time can drastically affect experimental results. Care must be taken to minimize DNA damage during the protocol and samples must be viable, as necrotic or apoptotic cells cannot be analyzed. While there has been some success in using cryopreserved cells for the comet assay, it is suggested that live cells are prepared immediately before use to ensure experimental quality and reproducibility. The number of cells able to be analyzed in 1 day is limited to 500–600 individual cells if performed by hand or 50–60 slides if using an automated software, reducing the utility of the comet assay for high throughput studies.

The comet assay may be performed under alkaline or neutral conditions. The neutral comet assay exhibits reduced sensitivity compared to the alkaline comet assay, but it detects solely double-stranded breaks, whereas the alkaline comet assay detects both single- and double-stranded DNA breaks. The principle of alkaline methods is that only if the DNA-base pairing is disrupted by an alkaline environment, the breaks can be detected. At neutral pH, however, the discontinuities in the single stranded breaks cannot be detected as the DNA is supercoiled and tightly packed, and in this condition only double stranded brakes will contribute to the formation of the comet. The methods for both alkaline and neutral comet assays are described herein.

Methods of Circulating and Tissue MDSC Test by FACS

Reagent Preparation

1. Chill lysis solution at 4°C for 20 min before use (see Note 2).
2. Prepare LMAgarose:
• Loosen the LMAgarose bottle cap and heat at 90–100°C in a water bath for 5 min, until agarose is molten.
• Place bottle in a 37–40°C water bath for 20 min to cool agarose before use (see Note 3).
3. Prepare alkaline solution (see Note 4):
• Combine 250 μL of 200 mM EDTA solution with 49.75 mL distilled water.
• Dissolve 0.6 g NaOH in the EDTA-dH₂O solution.
• Allow the alkaline solution to cool to room temperature before use.
4. Prepare alkaline electrophoresis solution:
• Dissolve 12 g NaOH in 2 mL of 500 mM EDTA disodium salt solution.
• Add distilled water to 1 L once the NaOH is dissolved.
5. Prepare 1XSYBR^® Green I staining solution by adding 1 μL SYBR^® Green I concentrate solution (10,000X) to 10 mL 1XTE buffer, pH 7.5.

Sample Preparation and Comet Assay

1. Aliquot 250 μL of molten LMAgarose into a microcentrifuge tube and place in a 37–40°C water bath for 20 min (see Note 5).
2. Count cells for the assay and suspend in ice cold 1XPBS (without Ca²⁺ and Mg²⁺) at a concentration of approximately 10⁵ cells/mL (see Note 6).
3. Combine 30 μL cell suspension (at a concentration of ~10⁵ cells/mL) with 250μL LMAgarose (cooled to 37°C). Immediately pipette 50 μL of cell/LMAgarose suspension onto a CometSlide™ (see Note 7).
4. Place the slides on a flat surface for 10 min at room temperature, then place the slides in the dark for 30 min at 4°C (see Note 8).
5. Incubate slides in chilled Lysis solution for 60 min at 4°C (see Note 9).
6. Tap off excess Lysis solution and immerse the slides in alkaline solution for 60 min at room temperature. Proceed with step 7 for the Alkaline Comet Assay or with step 8 for the Neutral Comet Assay.
7. For the Alkaline Comet Assay:
• Place the slides flat on a gel tray aligned equidistant from the electrodes.
• Carefully pour the alkaline solution into the tray until it is just covering the slides.
• Set the voltage to 1.15 V/cm and add or remove alkaline buffer until the current is approximately 300 mA. Perform electrophoresis for 60 min (see Note 10).
• Tap off excess electrophoresis solution and rinse the slides three times in distilled water.
• Continue with step 9.
8. For the Neutral Comet Assay:
• Tap off excess alkaline solution and wash the slides two times in 50 mL of 1X TBE buffer for 5 min each.
• Place the slide flat on a gel tray aligned equidistant from the electrodes.
• Carefully pour 1XTBE buffer into the tray until it is just covering the slides.
• Set the voltage to 1 V/cm and apply voltage for 45 min at room temperature.
• Continue with step 9.
9. Immerse the slides in 70% ethanol for 5 min (see Note 11).
10. Air dry samples overnight in the dark at room temperature.
11. Pipette 50 μL of 1X SYBR^® Green I staining solution onto each circle of dried agarose and air dry in the dark at room temperature for 1–2 h.
12. Score slides by epifluorescence microscopy. SYBR^® Green I maximum excitation and emission are 494 nm and 521 nm, respectively. Comets are scored by the length of their tail and can be scored manually or using commercially available software (see Note 12).

Browse by Primary Antibodies

Browse by Secondary Antibodies

Browse by Control Antibodies