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Cometassay reagent kit for single cell gel electrophoresis assay

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The CometAssay Reagent Kit is a laboratory tool used for the single cell gel electrophoresis assay, also known as the Comet assay. This kit provides the necessary reagents to perform this DNA damage assessment technique in a cellular sample.

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Lab products found in correlation

Sybr gold, by Thermo Fisher Scientific (3 mentions) Sub cell gt electrophoresis system, by Bio-Rad (2 mentions) Dpbs ca2 and mg2 free, by Thermo Fisher Scientific (2 mentions) Sybr green nucleic acid stain, by Thermo Fisher Scientific (2 mentions) Dmi 400b inverted microscope, by Leica (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Penicillin streptomycin antibiotic mixture, by Thermo Fisher Scientific (1 mentions) Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions) Milli q water purification system, by Merck Group (1 mentions) Ax70 fluorescence microscope, by Olympus (1 mentions) Doc cam hr ccd camera, by Molecular Devices (1 mentions) Bx53 microscope, by Olympus (1 mentions) Sn 38, by Selleck Chemicals (1 mentions) Dm 6000 power mosaic microscope, by Leica (1 mentions)

Close spelling variants of this product

Reagent Kit for Single Cell Gel Electrophoresis Assay CometAssay single cell gel electrophoresis assay CometAssay® single cell gel electrophoresis kit Single Cell Gel Electrophoresis Assay Kit CometAssay Reagent Kit CometAssay

19 protocols using cometassay reagent kit for single cell gel electrophoresis assay

1

DNA Damage Evaluation via Comet Assay

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The DNA damage was evaluated by using the Reagent Kit for Single Cell Gel Electrophoresis Assay/CometAssay (Trevigen), according to the manufacturer's instructions. The slides were viewed by using epifluorescence microscopy. The tail moment was calculated from 100 cells collected per single measurement by utilizing specialized comet software included in the Automated Comet Assay System (Loats Associates Inc).
Dai L., Qiao J., Nguyen D., Struckhoff A.P., Doyle L., Bonstaff K., Del Valle L., Parsons C., Toole B.P., Renne R, & Qin Z. (2016). Role of heme oxygenase-1 in the pathogenesis and tumorigenicity of Kaposi's sarcoma-associated herpesvirus. Oncotarget, 7(9), 10459-10471.
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2

DNA Damage Evaluation via CometAssay

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The DNA damage was evaluated by using the Reagent Kit for Single Cell Gel Electrophoresis Assay/CometAssay (Trevigen) as previously described 8 (link). The slides were viewed by using epifluorescence microscopy. The tail moment was calculated from 100 cells collected per single measurement by utilizing specialized comet software included in the Automated CometAssay System (Loats Associates Inc).
Dai L., Chen J., Cao Y., Del Valle L, & Qin Z. (2018). Ribonucleotide Reductase Inhibitor 3-AP Induces Oncogenic Virus Infected Cell Death and Represses Tumor Growth. Journal of Cancer, 9(23), 4503-4509.
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3

Comprehensive Cellular Assay Protocol

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SeC, AF, propidium iodide (PI), solid JC-1, DAPI, 2′,7′-dichlorofluorescein diacetate, MTT, bicinchoninic acid kit for protein determination were purchased from Sigma (St. Louis, MO, USA). Reagent kit for single-cell gel electrophoresis assay (Comet Assay) was purchased from Trevigen (Gaithersburg, MD, USA). TrxR1 Assay Kit was bought from Cayman (Ann Arbor, MI, USA). Dulbecco's modified Eagle's medium, fetal bovine serum, and the antibiotic mixture (penicillin-streptomycin) were purchased from Invitrogen (Carlsbad, CA, USA). Caspase-3 substrate (Ac-DEVD-AMC), caspase-9 substrate (Ac-LEHD-AFC), and caspase-8 substrate (IETD-AFC) were purchased from Calbiochem (San Diego, CA, USA). U0126 and LY294002 were obtained from Calbiochem. All of the antibodies used in this study were purchased from Cell Signaling Technology (Beverly, MA, USA). All of the solvents used were of high-performance liquid chromatography grade. The water used for all experiments was supplied by a Milli-Q water purification system from Millipore (Billerica, MA, USA).
Fan C., Zheng W., Fu X., Li X., Wong Y.S, & Chen T. (2014). Enhancement of auranofin-induced lung cancer cell apoptosis by selenocystine, a natural inhibitor of TrxR1 in vitro and in vivo. Cell Death & Disease, 5(4), e1191-.
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4

DNA Damage Assessment via Comet Assay

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The degree of the DNA damage was analyzed by means of the DNA-comet assay with CometAssay™ Reagent Kit for Single Cell Gel Electrophoresis Assay (Trevigen, Inc. 8405 Helgerman Ct. Gaithersburg, MD 20877). Average comet tails were selected for the calculation. One hundred nuclei per each slide were analyzed. The software package CometScore v. 1.5 (supplied by “TriTek Corp.” http://tritekcorp.com) was used for the comet assay performance.
Malinovskaya E.M., Ershova E.S., Okorokova N.A., Veiko V.P., Konkova M.S., Kozhina E.A., Savinova E.A., Porokhovnik L.N., Kutsev S.I., Veiko N.N, & Kostyuk S.V. (2019). Ribosomal DNA as DAMPs Signal for MCF7 Cancer Cells. Frontiers in Oncology, 9, 445.
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5

Alkaline Comet Assay for DNA Damage

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The alkaline comet assay was performed using CometAssay Reagent kit for Single Cell Gel Electrophoresis Assay (Trevigen, USA) according to the manufacturer’s protocol. Briefly, cells were collected then suspended at 1 × 105 cells per mL, and 20 μL cell suspension combined with 200 μL LMAgarose with final volume of 50 μL were then pipetted onto each CometSlide. When a clear ring appeared, the slides were incubated in Lysis Solution for 30 min. Then the slides were sunk in alkaline unwinding solution for 20 min at room temperature. Slides were electrophoresed in alkaline electrophoresis solution at 21 V, 300 mA for 30 min at 4 °C. The slides were drained and stained with 1× SYBR Gold (ThermoFisher). Cell images were captured using an Olympus AX70 fluorescence microscope (Olympus, Japan) and single cells tail length, tail DNA percentage were measured by CometScore version 2.0 (TriTek, USA) software [25 (link)].
Lv C., Fu S., Dong Q., Yu Z., Zhang G., Kong C., Fu C, & Zeng Y. (2019). PAGE4 promotes prostate cancer cells survive under oxidative stress through modulating MAPK/JNK/ERK pathway. Journal of Experimental & Clinical Cancer Research : CR, 38, 24.
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6

Neutral Comet Assay for DNA Damage

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The neutral comet assay was performed using the CometAssay Reagent kit for Single Cell Gel Electrophoresis Assay (Trevigen) in accordance with the manufacturer’s instructions. Electrophoresis was performed at 4°C, and slides were stained with PI and imaged on LeicaDMI8 microscope at 20×. Comet tail moments were obtained using an ImageJ plugin as previously described (Mathew et al., 2014 (link)). At least 50 cells per sample were analyzed from each independent experiment.
Chang E.Y., Novoa C.A., Aristizabal M.J., Coulombe Y., Segovia R., Chaturvedi R., Shen Y., Keong C., Tam A.S., Jones S.J., Masson J.Y., Kobor M.S, & Stirling P.C. (2017). RECQ-like helicases Sgs1 and BLM regulate R-loop–associated genome instability. The Journal of Cell Biology, 216(12), 3991-4005.
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7

Neutral Comet Assay Protocol

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Neutral comet assay was performed as previously described[22 (link)] using the CometAssay Reagent Kit for Single Cell Gel Electrophoresis Assay (Trevigen, 4250-050-K) in accordance with the manufacturer’s instructions.
Tsai S., Fournier L.A., Chang E.Y., Wells J.P., Minaker S.W., Zhu Y.D., Wang A.Y., Wang Y., Huntsman D.G, & Stirling P.C. (2021). ARID1A regulates R-loop associated DNA replication stress. PLoS Genetics, 17(4), e1009238.
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8

DNA Damage Quantification by Comet Assay

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DNA damage was assessed by using the CometAssay® reagent kit for single cell gel electrophoresis assay (Trevigen, MD USA), following the recommended protocol for neutral conditions, and adapting the gel electrophoresis methods for use in the Sub-Cell GT electrophoresis system (Bio-Rad, CA USA). Briefly, cells were collected from coverslips by treatment with 0.25% trypsin, pelleted and resuspended at 100,000 cells/ml in 1X DPBS (Ca2+ and Mg2+ free; Thermo Fisher Scientific) and verified to be greater than 95% viable by tryptan blue exclusion using an automated cell counter before continuing analysis. Aproximately 5,000 cells were embedded in low melting agarose, plated on slides and lysed overnight. The next day, electrophoresis was run at 30 Volts for 30 minutes in 1X TBE (National Diagnostics). Samples were fixed in 70% ethanol for 5 minutes, and slides were immersed in 1X TE buffer pH 8.0 (Ambion) with 1:10 of 10,000x SYBR green nucleic acid stain (Thermo Fisher Scientific). Fluorescent images were captured using a Leica DMI 400B inverted microscope for scoring.
Victor M.B., Richner M., Olsen H.E., Lee S.W., Monteys A.M., Ma C., Huh C.J., Zhang B., Davidson B.L., Yang X.W, & Yoo A.S. (2018). Striatal neurons directly converted from Huntington’s disease patient fibroblasts recapitulate age-associated disease phenotypes. Nature neuroscience, 21(3), 341-352.
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9

Comet Assay Protocol for Plant and Mammalian Cells

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A CometAssay® Reagent Kit for Single Cell Gel Electrophoresis Assay (Trevigen, Gaithersburg, MD, USA) was used to perform a comet assay on plant cells as described previously52 (link). For mammalian cultured cells, cells on 6-well plates were washed twice with PBS. After removing the PBS, 100 μl trypsin was added to the cells and incubated for 5 min at 37 °C. The trypsin reaction was stopped by adding 1 ml of 10% (v/v) FBS containing DMEM. Then, the cell mixture was transferred to a 1.5-ml tube. The cells were harvested by centrifugation at 800g for 1 min and resuspended in 1 ml PBS. Slides were prepared following the manufacturer’s protocol. Images of nuclei were obtained by inverted fluorescence microscopy on a BX53 microscope (Olympus, Shinjuku-ku, Tokyo, Japan) equipped with a DOC-CAM HR CCD camera (Molecular Devices, Chuo-ku, Tokyo, Japan). Images were analyzed using the ImageJ software plugin CometAssay distributed by the University of North Carolina School of Medicine, USA (https://www.med.unc.edu/microscopy/resources/imagej-plugins-and-macros/comet-assay).
Sakamoto T., Tsujimoto-Inui Y., Sotta N., Hirakawa T., Matsunaga T.M., Fukao Y., Matsunaga S, & Fujiwara T. (2018). Proteasomal degradation of BRAHMA promotes Boron tolerance in Arabidopsis. Nature Communications, 9, 5285.
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10

Neutral Comet Assay for DNA Damage

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The neutral comet assay was performed using the CometAssay Reagent Kit for Single Cell Gel Electrophoresis Assay (Trevigen) in accordance with the manufacturer’s instructions. In brief, cells were combined with LMAgarose at 37°C at a ratio of 1:10 (cell:agarose) and spread onto CometSlide. After gelling at 4°C in the dark, the slides were then immersed in 4°C Lysis Solution overnight. The next day, slides were removed from Lysis Solution and immersed in 4°C 1 × Neutral Electrophoresis Buffer for 30 min. Electrophoresis was then performed at 4°C at 20 Volts for 30 min. Slides were then immersed in DNA Precipitation Solution for 30 min followed by 70% ethanol for 30 min at room temperature, and dried at 37°C for 10 min. Finally, slides were stained with PI and imaged on LeicaDMI8 microscope at ×20. Comet tail moments were obtained using an ImageJ plugin as previously described [36 (link)]. At least 50 cells per sample were analyzed from each independent experiment.
Wells J.P., Chang E.Y., Dinatto L., White J., Ryall S, & Stirling P.C. (2022). RAD18 opposes transcription-associated genome instability through FANCD2 recruitment. PLOS Genetics, 18(12), e1010309.
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