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Index / Trevigen / CometAssay Control Cells / Product Detail : 4256-010-CC CometAssay Control Cells
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Product name : CometAssay Control Cells

Catalog number : 4256-010-CC

Quantity: 10 assays

Availability: Yes

Supplier name : Trevigen

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CometAssay® Control Cells
For Single Cell Gel Electrophoresis Assay
Catalog # 4256-010-CC

Sufficient materials for 10 assays.

The Assay Protocol described below is written as a Quick Reference using alkaline Comet Control Cells (cat#4256-010-CC). Reagents and detailed instructions including reagent preparation are provided with Trevigen’s CometAssay® Kits (Please See Section XI).
This page is designed to be photocopied and used as a checklist:
1. Chill Lysis Solution at 4°C for at least 20 minutes before use.
2. Melt LMAgarose and cool in a 37°C water bath for at least 20 minutes.
3. Combine 50 μl of CCO (control cells) with 500 μl molten LMAgarose(at 37˚C) and immediately spread 50 μl per well over a 2 well, or 30 ul per well for a 20 well CometSlide™.
4. Repeat step 3 for samples CC1, CC2, and CC3, respectively.
5. Place slides flat at 4°C in the dark for 10 minutes.
6. Immerse slides in prechilled Lysis Solution at 4°C, for 30 minutes.
7. Immerse slides in 50 ml freshly prepared Alkaline Unwinding Solution, pH>13 for 20 minutes at room temperature, in the dark.
8. For the CometAssay® ES tank, add 950 ml prechilled Alkaline Electrophoresis Solution, place slides in electrophoresis slide tray and cover with Slide Tray Overlay. Set power supply to 21 volts and apply voltage for 30 minutes. For other horizontal electrophoresis units, carefully pour cold Alkaline Solution until level just covers samples (not to exceed 0.5 cm). Set the voltage to 1 Volt/cm. Add or remove buffer to adjust current to ~220 mA.
9. Immerse slides twice in dH2O for 5 minutes each, then in 70% ethanol for 5 minutes.
10. Dry samples at ≤ 45°C for 10-15 minutes.
11. Place 100 μl (2 well slide) or 50 μl (20 well slide) of diluted SYBR® Green I onto each sample for 30 minutes. Remove excess SYBR solution. Allow slide to dry completely at room temperature in the dark.
12. View slide by epifluorescence microscopy. (SYBR® Green I1 has excitation and emission wavelengths of 425 nm and 521 nm, respectively. A fluorescein filter is adequate.)
1 SYBR® Green I is a registered product of Molecular Probes, Eugene OR, and is sold under license from Molecular Probes, Inc. Please see p.9 for complete licensing terms. Use of this reagent outside of the scope of these terms is not endorsed by Trevigen,Inc.

II. Background
Trevigen’s CometAssay®, or single cell gel electrophoresis assay, provides a simple and effective method for evaluating DNA damage in cells. The principle of the assay is based upon the ability of denatured, cleaved DNA fragments to migrate out of the nucleoid under the influence of an electric field, whereas undamaged DNA migrates slower and remains within the confines of the nucleoid when a current is applied. Evaluation of the DNA “comet” tail shape and migration pattern allows for assessment of DNA damage. The Alkaline CometAssay® allows for sensitive detection of both single and double-stranded breaks. In this assay, cells are immobilized in a bed of low melting point agarose, on a Trevigen CometSlide™. Following gentle cell lysis, samples are treated with alkali to unwind and denature the DNA and hydrolyze sites of DNA damage. The samples are then submitted to electrophoresis and staining with a fluorescent DNA intercalating dye. The sample is then visualized by epifluorescence microscopy. Quantitative and statistical data can readily be generated by analysis of the results using one of several commercially available image analysis software packages which calculate tail length, percent DNA in the tail and tail moment.
Trevigen has developed a set of suspension cell preparations containing different levels of DNA damage to be used as controls with Trevigen’s CometAssay® Kits. When performing alkaline electrophoresis, the four control cell populations show incremental increases in percent DNA in the tail. The healthy control cell population (CC0) was treated with Etoposide under various conditions to increase the amount of damage in populations CC1, CC2 and CC3, respectively. Etoposide is a DNA synthesis inhibitor that induces doublestranded and single-stranded DNA breaks. These cryopreserved controls are designed to act as controls to standardize and compare alkaline electrophoresis methods between individual users and laboratories.

III. Precautions and Limitations
1. For Research Use Only. Not for use in diagnostic procedures.
2. The physical, chemical, and toxicological properties of the CometAssay® Control Cells may not have been fully investigated. Therefore, Trevigen recommends the use of gloves, lab coats, and eye protection while using any of these chemical reagents. Trevigen assumes no liability for damage resulting from handling or contact with these products.

IV. Materials Supplied
V. Materials/Equipment Required But Not Supplied Equipment:
1. 20–200 μl, 200–1,000 μl pipettors, and tips
2. Table Top Centrifuge (vertical rotor)
3. Water Bath
4. -80°C Freezer
5. Liquid Nitrogen Storage System
6. CometAssay® ES (cat# 4250-050-ES) or other horizontal electrophoresis unit.
1. Ice cold 1X PBS pH 7.4, Ca++ and Mg++ free
2. Isopropanol
3. NaOH Pellets
4. 0.5 M EDTA (pH 8.0)
5. CometAssay® Kit1 (required)
1 Available from Trevigen; refer to Section XI. for ordering information.

VI. Preparation of Control Cells
Control cells should be prepared immediately before starting the CometAssay® protocol.
CometAssay® Control Cells are stored using a Liquid Nitrogen Storage System. To avoid the accumulation of damage due to freeze thaw, the control cells should be aliquotted and cryopreserved as described below.
1. Recover cells by submerging in 37°C water bath to quickly thaw cells, and place on ice.
2. Gently invert to mix and transfer 50 μl aliquots into freezing vials.
3. Freeze at -80°C with -1°C per minute freezing rate. This can be done by placing the vials in a Styrofoam box containing room temperature Isopropanol and placing in a -80°C freezer overnight. Vials are placed in a plastic box or rack then placed in room temperature isopropanol. The lid of the Styrofoam container is put in place then the box is placed in the -80°C freezer.
4. Transfer to Liquid Nitrogen System for long-term storage.

Assay Preparation Protocol:
1. Remove 50 μl aliquots of CC0, CC1, CC2 and CC3 CometAssay® Control Cells from Liquid Nitrogen Storage.
2. Quickly thaw cells by submerging in 37°C water bath, and add 600 μl of ice cold 1X PBS (Ca++ and Mg++ free).
3. Centrifuge cells at 150 x g for 5 minutes and gently remove supernatant, except for about 50 μl.
1) A cell pellet will not be visible after centrifugation.
2) Removing supernatant completely will result in cell loss.
4. Gently resuspend cell pellet in 50 μl of ice cold 1X PBS.
5. Immediately use the cells in the CometAssay® protocol described for Alkaline

VII. Assay Protocol
The assay protocol is the same as listed in the checklist on page 1. Additional reagents are required. For information regarding preparation of all needed reagents, please see the instructions for use for Trevigen’s CometAssay® (cat# 4250-050-K).

VIII. Data Interpretation
When excited (425–521 nm) the DNA-bound SYBR® Green I emits green light. In healthy cells the fluorescence is confined to the nucleoid (comprised of high molecular weight DNA): undamaged DNA is supercoiled and thus, does not migrate very far out of the nucleoid under the influence of an electric current. Whereas in cells that have accrued DNA damage, migrating fragments (comet tail) from the nucleoid (comet head) are observed. The negatively charged DNA migrates toward the anode and the extrusion length reflects increasing relaxation of supercoiling, which is indicative of damage. Common descriptors of DNA damage for alkaline comet assays are Percent DNA in the Tail, and Tail Moment. Percent DNA in the Tail is a normalized measure of the percent of total cell DNA found in the tail. Tail moment is a damage measure combining the amount of DNA in the tail with distance of migration. In neutral comet assays, Tail Moment is primarily used, since tail length continues to increase in contrast to alkaline comet tails which have finite lengths. Qualitative Analysis (Alkaline CometAssay®) The comet tail can be scored according to DNA content (intensity). 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 high intensity tail DNA content. At least 50 cells should be scored per sample. Quantitative Analysis (Alkaline CometAssay®) There are several image analysis systems that are suitable for quantitation of CometAssay® data. The more sophisticated systems include the microscope, camera and computer analysis package. These systems can be set up to measure the length of DNA migration, image length, nuclear size, and calculate DNA damage parameters. At least 50 randomly selected cells should be analyzed per sample. A list of commercially available software package is available from Trevigen.

Featured Data:
To evaluate the degree of damage, the Control Cells were processed using the CometAssay® System under defined electrophoresis conditions. In the example below, alkaline electrophoresis was performed on two-well slides (4250-050-03) using CometAssay® Kit (4250-050-K). Images were captured and analyzed using Loats Associates, Inc Comet Analysis System. Data was exported into Analyze-it™ ( for Microsoft Excel to graphically represent the spread of data. In Figure 1a, data collected from 50 cells for each Control Cell population (lot# 12161M6) are shown as side-by side vertical box plots for comparison. The diamond shows the mean and confidence interval around the mean. The notched box shows the median, lower and upper quartiles, and the 75% confidence interval around the median. For each lot of Control Cells, population values are provided in a data sheet. An example is provided below.

X. References
1. Lemay, M. and K.A. Wood, 1999. Detection of DNA damage and identification of UVinduced photoproducts using the CometAssay® kit. BioTechniques 27(4):846-51.
2. Angelis, K.J., M. Dusinska and A.R. Collins. 1999. Single cell gel electrophoresis: Detection of DNA damage at different levels of sensitivity. Electrophoresis 20:2133-8.
3. Morris, E.J., J.C. Dreixler, K-Y. Cheng, P.M. Wilson, R.M. Gin and H.M. Geller. 1999. Optimization of single-cell gel electrophoresis (SCGE) for quantitative analysis of neuronal DNA damage. BioTechniques 26:282-9.
4. Malyapa, R.S., C. Bi, E.W. Ahern, and J.L. Roti, 1998. Detection of DNA damage by the alkali comet assay after exposure to low dose gamma radiation. Radiation Res 149:396- 400.
5. Henderson, L., A. Wolfreys, J. Fedyk, C. Bourner and S. Windeback, 1998. The ability for the comet assay to discriminate between genotoxins and cytotoxins. Mutagenesis 13:89-94.
6. Visvardis, E.E., A.M. Tassiou and S.M. Piperakis,1997. Study of DNA damage induction and repair capacity of fresh cryopreserved lymphocytes exposed to H2O2 and γ- irradiation with the alkaline comet assay. Mutation Res 383:71-80.
7. Fairbairn, D.W., P.L. Olive and K.L. O'Neill. 1995. The comet assay: a comprehensive review. Mutation Res 339:37-59.
8. Collins, A.R., A.G. Ma and S.J. Duthie, 1995. The kinetics of repair of oxidative DNA damage (strand breaks and oxidized pyrimidine dimers) in human cells. Mutation Res
9. Singh, N.P., M.T. McCoy, R.R. Tice and E.L. Schneider, 1988. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175:184-91.
10. Östling, O. and K. J. Johanson, 1984. Microelectrophoretic study of radiation- induced DNA damage in individual cells. Biochem Biophys Res Commun 123:291-8.

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