Comet assay kit

Manufactured by Cell Biolabs

Sourced in United States

The Comet assay kit is a laboratory tool used to detect and measure DNA damage at the single-cell level. It provides a quantitative analysis of DNA fragmentation, which is an indicator of cellular stress or genotoxicity.

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Lab products found in correlation

Fsx100 microscope, by Olympus (2 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions) Axiovert 40 cfl, by Zeiss (1 mentions) Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions) Transdetect in situ fluorescein tunel cell apoptosis detection kit, by Transgene (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Protein extraction kit, by Keygen Biotech (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions) Sodium dodecyl sulfate (sds), by Merck Group (1 mentions) Bca protein assay kit, by Beyotime (1 mentions) Methyl thiazolyl tetrazolium (mtt), by Roche (1 mentions) Streptomycin, by Keygen Biotech (1 mentions) Penicillin, by Keygen Biotech (1 mentions) Fluorescence microscope, by Olympus (1 mentions) Thunder imaging system, by Leica (1 mentions)

12 protocols using comet assay kit

Comet Assay for DNA Damage Analysis

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The DNA damage induced by X-ray irradiation was analyzed via SCGE using a comet assay kit (Cell Biolabs, Inc.) at high pH. The cells were mixed with molten agarose. The DNA was relaxed and denatured using a lysis buffer and an alkaline solution. After the electrophoresis of these samples, they were dried and stained, and then observed using epifluorescence microscopy. The damaged DNA was found to migrate further than the intact DNA and then form a comet-shaped structure.
The degree of DNA damage was evaluated by measuring the displacement between the genetic material of the “comet head” (the nucleus of the cell) and the resulting “comet tail” (the DNA that has escaped from the nucleus). For every sample, we assessed the DNA damage for 50 cells. The tail moment is a typical index that considers both the migration of the genetic material and the relative amount of DNA in the tail. The % of DNA in the tail is calculated thus:

% tail DNA (TDNA) = 100 \times tail DNA intensity / cell DNA intensity

The tail moment is calculated thus, based on the formula supplied with the Cell Biolabs comet assay kit:

Tail moment = TDNA \times length of tail

The data were analyzed using CometScore (TriTek Corp.).

Li H., Cui Y., Li F., Shi W., Gao W., Wang X, & Zeng Q. (2018). Measuring the lactate-to-creatine ratio via 1H NMR spectroscopy can be used to noninvasively evaluate apoptosis in glioma cells after X-ray irradiation. Cellular & Molecular Biology Letters, 23, 27.

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DNA Double-Strand Break Analysis in ESCs

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The presence of DNA DSBs was analyzed using the Comet Assay kit from Cell Biolabs according to the manufacturer’s instructions. In brief, Sall4^+/− and Sall4^−/− ESCs plated on gelatin-coated dishes were treated with DOX for 4 h, harvested, and washed with ice-cold PBS. Cells were resuspended at a density of 10⁵ cells/ml, mixed with molten agarose at 1:10 ratio, spread onto 3-well Comet Assay slides, and solidified for 15 min at 4°C. Slides were immersed in lysis solution and electrophoresed in chilled TBE (Tris-borate-EDTA) buffer for 15 min at 20 V. Slides were then fixed in 70% ethanol and dried, and DNA was labeled with Vista Green DNA Dye. Images were captured at 10× magnification with an inverted microscope (Axiovert 40 CFL; Carl Zeiss) and analyzed using Comet Assay IV software (Perceptive Instruments); at least 100 cells were analyzed per sample.

Xiong J., Todorova D., Su N.Y., Kim J., Lee P.J., Shen Z., Briggs S.P, & Xu Y. (2015). Stemness factor Sall4 is required for DNA damage response in embryonic stem cells. The Journal of Cell Biology, 208(5), 513-520.

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Dioscin-Induced Apoptosis Pathway

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Dioscin was obtained from the Chengdu Research Institute of Biology of the Chinese Academy of Sciences. MTT was obtained from Roche Diagnostics. The Protein Extraction kit, penicillin and streptomycin were purchased from Nanjing KeyGen Biotech Co., Ltd. The BCA protein assay kit was obtained from Beyotime Institute of Biotechnology. Carboxymethylcellulose-Na, Tris and SDS were purchased from Sigma-Aldrich (Merck KGaA). The Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco (Thermo Fisher Scientific, Inc.). DAPI was purchased from Sigma-Aldrich (Merck KGaA). The comet assay kit was purchased from Cell Biolabs, Inc. The terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay was performed using the TransDetect^®In situ Fluorescein TUNEL Cell Apoptosis Detection kit (TransGen Biotech Co., Ltd.). Primary antibodies were purchased from ProteinTech Group, Inc., and Wuhan Boster Biological Technology, Ltd. (Table SI). Secondary antibodies were purchased from ProteinTech Group, Inc. Lipofectamine^® 2000 was purchased from Thermo Fisher Scientific, Inc., and p53 small interfering (si)RNAs were purchased from Guangzhou RiboBio Co., Ltd. Z-VAD-FMK/pan-caspase inhibitor was purchased from MedChemExpress.

Wang P., Wang C, & Liu C. (2020). Antitumor effects of dioscin in A431 cells via adjusting ATM/p53-mediated cell apoptosis, DNA damage and migration. Oncology Letters, 21(1), 59.

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Dioscin-Induced DNA Damage Quantification

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The extent of DNA damage was determined using the Comet assay kit according to the manufacturer's protocol (Cell Biolabs, Inc.). A total of 2×10⁵ cells/well of A431 cells were incubated in 6-well plates. After being treated with different concentrations of dioscin (2.9, 5.8 and 11.6 µM) at 37°C for 24 h, cell images were captured using a fluorescence microscope at ×200 magnification (Olympus Corporation). Finally, the Comet Assay Software Project v1.2.2 (CaspLab) was used to analyze the selected cells (50 cells from each of the two replicate slides).

Wang P., Wang C, & Liu C. (2020). Antitumor effects of dioscin in A431 cells via adjusting ATM/p53-mediated cell apoptosis, DNA damage and migration. Oncology Letters, 21(1), 59.

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Comet Assay for DNA Damage Quantification

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Comet Assay Kit (Cell Biolabs) used according to manufacturers’ protocol. Briefly, 1% low-gel temperature agarose melted and kept in a molten state at 37°C. 75μL agarose pipeted onto slide wells and chilled 15 min at 4°C to create a base layer. Cells were harvested, washed with chilled PBS, and resuspended to 10⁵ cells/mL in chilled PBS. Cells were then mixed 1:10 with molten agarose, 75μL pipetted on top of the base layer, and chilled for 15 min at 4°C. Slides were then submerged 60min in chilled lysis buffer, 30min in chilled alkaline solution, then electrophoresed in chilled alkaline electrophoresis solution for 30 min at 1 V/cm at 300mA. Slides were washed three times with chilled d.i. H₂O, fixed with cold 70% ethanol for 5 min, and air dried. Slides were stained with 100μL/well of 0.1mg/mL propidium iodide and incubated at least 15 min then imaged on a Thunder Imaging System (Leica). Comet tail moment was quantified using the ImageJ plugin OpenComet^{61 (link)}; poorly fit comets were manually excluded. 47–126 cells per treatment counted for THP-1, and 9–40 for MOLM-13.

Brabson J.P., Leesang T., Yap Y.S., Wang J., Lam M.Q., Fang B., Dolgalev I., Barbieri D.A., Strippoli V., Bañuelos C.P., Mohammad S., Lyon P., Chaudhry S., Donich D., Swirski A., Roberts E., Diaz I., Karl D., Santos H.G., Shiekhattar R., Neel B.G., Nimer S.D., Verdun R.E., Bilbao D., Figueroa M.E, & Cimmino L. (2023). Oxidized mC modulates synthetic lethality to PARP inhibitors for the treatment of leukemia. Cell reports, 42(1), 112027.

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Comet Assay for DNA Damage

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Cells were collected at indicated time points and re-suspended in low-melting-point agarose provided by Cell Biolabs’s COMET Assay kit (STA-351, Cell Biolabs). The assay was run per the manufacture’s protocol. Images were taken using an Olympus FSX-100 microscope and quantitated using the OpenComet plugin for ImageJ.

Madhav A., Andres A., Duong F., Mishra R., Haldar S., Liu Z., Angara B., Gottlieb R., Zumsteg Z.S, & Bhowmick N.A. (2018). Antagonizing CD105 enhances radiation sensitivity in prostate cancer. Oncogene, 37(32), 4385-4397.

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Comet Assay for DNA Damage

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DNA damage was measured by the Comet assay kit (Cell Biolabs, STA-355, San Diego, CA, USA), according to the manufacturer’s instructions. The percentage of tail DNA was quantified according to a previous study [70 (link)].

Su B.C., Pan C.Y, & Chen J.Y. (2019). Antimicrobial Peptide TP4 Induces ROS-Mediated Necrosis by Triggering Mitochondrial Dysfunction in Wild-Type and Mutant p53 Glioblastoma Cells. Cancers, 11(2), 171.

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Comet Assay of Aconitine Cytotoxicity

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The culture and treatment of A2780 cells were the same as TUNEL staining. After the treatment with different concentrations of aconitine (100, 200 and 400 µg/ml) for 24 h, the extent of DNA damage was determined by the Comet assay kit (Cell Biolabs, Inc.), according to the manufacturer's instructions. Images were taken using a fluorescence microscope and the Comet Assay Software Project (CASP) 1.2.2 (CaspLab) was used to analyze 50 cells from each of the 2 replicate slides.

Wang X., Lin Y, & Zheng Y. (2020). Antitumor effects of aconitine in A2780 cells via estrogen receptor β-mediated apoptosis, DNA damage and migration. Molecular Medicine Reports, 22(3), 2318-2328.

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Alkaline Comet Assay for DNA Damage

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Alkaline comet assays were performed using a comet assay kit (Cell Biolabs, San Diego, CA, USA), according to the manufacturer's protocol. The cells were mixed with low-melting agarose gel and spread on the comet slide. Subsequently, the cells were lysed on ice with the lysis solution and then equilibrated with an alkaline electrophoresis buffer. Images of the slides were obtained using a fluorescence microscope. The tail streaks of 50 randomly selected cells were measured and averaged.

Song I.S., Jeong Y.J., Jung Y., Park Y.H., Shim S., Kim S.J., Eom D.W., Hong S.M., Lee P.C., Kim S.U, & Jang S.W. (2021). The sulfiredoxin-peroxiredoxin redox system regulates the stemness and survival of colon cancer stem cells. Redox Biology, 48, 102190.

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Measuring Oxidative DNA Damage in Cells

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Thiobarbituric acid-reactive substances (TBARS) were used for measurement of lipid peroxidation. Cell extracts were mixed with 1 mL TBA solution [0.375% thiobarbituric acid in 0.25 N HCl containing 15% (w/w) trichloroacetic acid] [21 (link)]. Lipid peroxidation was also detected by using a fluorescent DPPP probe [24 (link)]. The levels of 8-hydroxy-2′-deoxyguanosine (8-OH-dG) in LLC cells were measured with a fluorescent binding assay as described previously [25 (link)]. Cells were fixed and permeabilized with ice-cold methanol for 15 min. DNA damage was visualized with avidin-conjugated TRITC (1 : 200 dilution) using a fluorescence microscope. The comet assay was also performed using the Comet Assay Kit (Cell Biolabs Inc., San Diego, CA). Cell extracts were washed with cold PBS and centrifuged. The cell pellet was mixed with Comet Agarose at 1 : 10 ratio (v/v) and pipetted onto the Comet assay slide. Slides were dried at 4°C in the dark for 15 min and incubated in chilled lysis solution at 4°C in the dark for another 15 min. After washing with TBE buffer (50 mM Tris, 50 mM boric acid, and 0.2 mM EDTA), the samples were subjected to electrophoresis and stained with Vista Green DNA Dye. Images were obtained with a microscope. The percentage of tail DNA of the cells in each slide was measured and quantified.

Park J.H., Ku H.J., Lee J.H, & Park J.W. (2017). Idh2 Deficiency Exacerbates Acrolein-Induced Lung Injury through Mitochondrial Redox Environment Deterioration. Oxidative Medicine and Cellular Longevity, 2017, 1595103.

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