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Cometassay electrophoresis system 2

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The CometAssay Electrophoresis System II is a laboratory instrument designed to perform comet assay analysis. The system provides the necessary components for the electrophoresis and analysis of DNA damage and repair in individual cells.

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Lab products found in correlation

Comet assay kit, by Bio-Techne (3 mentions) Sybr gold, by Thermo Fisher Scientific (3 mentions) Lsm 780, by Zeiss (2 mentions) Cometslide, by Bio-Techne (2 mentions) Axioskop 2 plus, by Zeiss (2 mentions) Lsm 700, by Zeiss (2 mentions) P chk2, by Cell Signaling Technology (1 mentions) P atm, by Cell Signaling Technology (1 mentions) P histone h3, by Cell Signaling Technology (1 mentions) Dp70 ccd camera, by Olympus (1 mentions) Ix70 inverted fluorescence microscope, by Olympus (1 mentions) Lysis solution, by Bio-Techne (1 mentions) Prolong diamond antifade mountant, by Thermo Fisher Scientific (1 mentions) Dimethyl sulfoxide (dmso), by Nacalai Tesque (1 mentions) Bz x810, by Keyence (1 mentions) Interferin, by Polyplus Transfection (1 mentions) Comet analysis software, by Bio-Techne (1 mentions) Evos fl cell imaging system, by Thermo Fisher Scientific (1 mentions) Lysis buffer, by Bio-Techne (1 mentions) Sybr gold nucleic acid gel stain, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

CometAssay (52 citations) CometAssay Electrophoresis System (14 citations)

9 protocols using cometassay electrophoresis system 2

1

Evaluating DNA Damage Response via IF and Comet

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Immunofluorescence (IF) was performed based on manufacturer instructions. Cells were: washed in PBS; fixed for 20 minutes at RT in 4% PFA; blocked for 1 hour at room temperature in 5% goat serum and 1% Triton X-100 in 1× PBS; incubated with primary antibody overnight at 4 degrees in 1% goat serum and 1% Triton X-100 in 1× PBS antibody diluent; incubated with secondary antibody in diluent for 1 hour at RT; and then mounted with DAPI-containing mounting media (cat#P36935). Primary antibodies used include pHistoneH3 (Cell Signaling; 1:500), pATM (cat#ab36810), pChk2 (cat#21997), MLH1 (cat#WH0004292M2). Cells were treated with fulvestrant for 48 hours before evaluation. Alkaline comet assay was performed as per the manufacturer’s instructions on a CometAssay Electrophoresis SystemII (Trevigen, cat#4250-050-ES). Calculation of nuclei with DNA damage was performed using CASPLab software27 to calculate the ratio of DNA content in tail/head. Cut-offs for categorization were set as 0–0.5, 0.5–1, 1–5 and >5 for No, Low, Med and High DNA damage. Fluorescent images were captured with a Nikon microscope and quantified with ImageJ.
Haricharan S., Punturi N., Singh P., Holloway K.R., Anurag M., Schmelz J., Schmidt C., Lei J.T., Suman V., Hunt K., Olson JA J.r., Hoog J., Li S., Huang S., Edwards D.P., Kavuri S.M., Bainbridge M.N., Ma C.X, & Ellis M.J. (2017). Loss of MutL disrupts Chk2-dependent cell cycle control through CDK4/6 to promote intrinsic endocrine therapy resistance in primary breast cancer. Cancer discovery, 7(10), 1168-1183.
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2

Detecting DNA Strand Breaks Using Comet Assay

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DNA strand breaks were detected using single cell gel electrophoresis under both neutral pH and alkaline conditions using CometAssay Electrophoresis System II (Trevigen, Cat. #4250-050-ES). After electrophoresis, cells embedded in low-melting agarose (comets) were fixed with 70% ethanol and stained with SYBR Gold (Thermo Fisher Scientific, Cat. #S-11494). Images of comets in random fields were captured under Olympus IX70 inverted fluorescence microscope using Olympus DP70 CCD camera and 4× objective, and subjected to quantitation using Comet Assay IV software (Instem). At least 200 cells per treatment condition were scored. An increase in Olive tail moment from a neutral comet assay was used as a readout for DSBs, whereas that from an alkaline comet assay for the sum of SSBs and DSBs. Additional images were taken using 10× or 20× objectives to feature more details of comets.
Liu X., Jiang Y., Takata K.I., Nowak B., Liu C., Wood R.D., Hittelman W.N, & Plunkett W. (2019). CNDAC-induced DNA double strand breaks cause aberrant mitosis prior to cell death. Molecular cancer therapeutics, 18(12), 2283-2295.
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3

Comet Assay for DNA Damage Assessment

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Comet assay was performed using CometAssay reagents (Trevigen) according to the manufacturer’s instructions. Briefly, cultured erythroblasts were mixed with the molten Comet LMAgarose, and plated onto the CometSlide. Slides were cooled at 4 °C for 30 min and then immersed in Lysis Solution (Trevigen) containing 10% DMSO (Nacalai Tesque) at 4 °C for 3 h. Then slides were immersed in Alkaline Unwinding Solution (200 mM NaOH and 1 mM EDTA, pH > 13) at 4 °C for 1 h. Next, alkaline electrophoresis was performed in a cooled Alkaline Electrophoresis Solution (200 mM NaOH, 1 mM EDTA, pH > 13) using cooled CometAssay Electrophoresis System II (Trevigen, 21 V, 30 min). Slides were washed twice in distilled water and once in 70% ethanol, air-dried, stained with SYBR Gold (Thermo Fisher Scientific) at room temperature for 30 min, and washed in distilled water. Slides were then air-dried completely and mounted with Prolong Diamond Antifade Mountant (Thermo Fisher Scientific). Images were acquired using the fluorescence microscope BZ-X810 (Keyence) and analyzed using CASPLab software (v1.2.2, https://casplab.com/)92 (link) with default settings.
Yoshinaga M., Han K., Morgens D.W., Horii T., Kobayashi R., Tsuruyama T., Hia F., Yasukura S., Kajiya A., Cai T., Cruz P.H., Vandenbon A., Suzuki Y., Kawahara Y., Hatada I., Bassik M.C, & Takeuchi O. (2022). The N6-methyladenosine methyltransferase METTL16 enables erythropoiesis through safeguarding genome integrity. Nature Communications, 13, 6435.
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4

Comet Assay for DNA Damage Evaluation

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U2OS cells were seeded in 6-well plates (1.0 × 105 cells/well). Twenty-four hours later, cells were transfected with non-targeting siRNA or MED1 pool siRNAs using INTERFERin (Polyplus Transfection). After another 24 h, cells were treated with X-rays and incubated for 24 h before harvesting. After washing with PBS, cells were re-suspended in PBS at a concentration of ~ 1.0 × 106 cells/ml. DNA damage was evaluated using the Comet Assay Kit (4250-050-K, Trevigen) following manufacturer’s instructions. Electrophoresis was run at 300 mA, 21 V for 30 min in electrophoresis buffer using the Comet Assay Electrophoresis System II (Trevigen). Slides were washed in neutralization buffer (0.4 M Tris–HCl pH 7.5) and counterstained with SYBR Gold (diluted 1:1000 in PBS) (Invitrogen). Images were acquired using a confocal microscope LSM780 (Carl Zeiss), and comet tail moment was estimated using CometScore software. At least 100 comets per sample were analyzed.
Honjoh H., Tanikawa M., Wada-Hiraike O., Oda K., Inaba H., Kukita A., Kawata Y., Kusakabe M., Tsuchimochi S., Taguchi A., Miyamoto Y., Sone K., Tsuruga T., Mori-Uchino M., Matsumoto Y, & Osuga Y. (2022). MED1, a novel binding partner of BRCA1, regulates homologous recombination and R-loop processing. Scientific Reports, 12, 17140.
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5

Comet Assay for DNA Damage Detection

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The Comet Assay Kit (Trevigen) was used according to the manufacturer's instructions. Briefly, cells were suspended in low-melt agarose, layered onto treated slides to promote attachment (500 cells per slide), lysed, and subjected to electrophoresis (1 V/cm) under alkaline conditions to reveal single- and double-stranded DNA breaks using the Comet Assay Electrophoresis System II (Trevigen). Samples were then fixed, dried, and stained with SYBR Gold. Images were acquired with a ×10× objective lens using the EVOS FL Cell Imaging System (Thermo Fisher). Comet tail size was quantified using Comet Analysis Software (Trevigen).
Barger C.J., Chee L., Albahrani M., Munoz-Trujillo C., Boghean L., Branick C., Odunsi K., Drapkin R., Zou L, & Karpf A.R. (2021). Co-regulation and function of FOXM1/RHNO1 bidirectional genes in cancer. eLife, 10, e55070.
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6

Comet Assay for Cellular DNA Damage

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Comet assay was performed following the manufacturer’s protocol (Trevigen). Briefly, after overnight incubation with gentamycin-supplemented medium, infected cells were collected using trypsin. Cells were mixed with LMAgarose (Trevigen) and spread on 20-well CometSlides (Trevigen). CometSlides were incubated in the lysis buffer (Trevigen) for 1 h at 4 °C to lyse cells, and then incubated in alkaline electrophoresis solution (deionized H2O containing 200 mM NaOH and 1 mM EDTA) for 20 min at room temperature. Electrophoresis was performed using the CometAssay Electrophoresis System II (Trevigen). DNA was stained with SYBR Gold nucleic acid gel stain (S-11494, Life Technologies) and visualized using the Leica DM6000B upright microscope.
Mousa J.J., Yang Y., Tomkovich S., Shima A., Newsome R.C., Tripathi P., Oswald E., Bruner S.D, & Jobin C. (2016). MATE transport of the E. coli-derived genotoxin colibactin. Nature microbiology, 1, 15009.
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7

Comet Assay for DNA Fragmentation

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Nuclear DNA fragmentation was monitored using a Comet Assay Kit and Comet Assay Electrophoresis System II (Trevigen, Gaithersburg, MD, USA) under alkaline and neutral conditions, according to the manufacturer’s instructions. H414 and HCT116 cells were transfected with control or ITPA siRNA. After incubation for 4 days, the cells were separately embedded in soft agarose on glass slides and then subjected to the assay. Comet images from more than 30 cells for each siRNA were captured using an Axioskop 2 plus equipped with an AxioCam (Carl Zeiss MicroImaging Japan, Tokyo, Japan), and were analysed using the Comet Assay Software Project (CASP) program73 (link) to quantify tail moment.
Yoneshima Y., Abolhassani N., Iyama T., Sakumi K., Shiomi N., Mori M., Shiomi T., Noda T., Tsuchimoto D, & Nakabeppu Y. (2016). Deoxyinosine triphosphate induces MLH1/PMS2- and p53-dependent cell growth arrest and DNA instability in mammalian cells. Scientific Reports, 6, 32849.
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8

Comet Assay for DNA Damage Assessment

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MMA+3 treated DN and DP cells were immobilized in low melting point agarose on a Trevigen CometSlide™ according to the Comet assay kit instructions. Immobilized cells were lysed with Lysis Solution with 10% DMSO overnight. On the next day, DNA in the lysed cells was unwound with basic pH buffer (8 g NaOH with 2 mL of 0.5 M EDTA in 1 L of deionized water, pH > 13) at RT for 45 min. After the unwinding step, slides were electrophoresed in ice cold basic buffer in CometAssay® Electrophoresis System II (Trevigen) at 21 V for 30 min. Slides were then washed, dried and stained with Sybr Gold (1:10000 dilution in TE buffer) and imaged using an epifluorescence microscope. Fifty randomly selected cells from each well were scored using CometScore software (TriTek Corp., Sumerduck, VA). DNA damage was reported by percentage of DNA in tail (Collins, 2004 (link)).
Xu H., Medina S., Lauer F.T., Douillet C., Liu K.J., Stýblo M, & Burchiel S.W. (2017). Genotoxicity induced by monomethylarsonous acid (MMA+3) in mouse thymic developing T cells. Toxicology letters, 279, 60-66.
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9

Comet Assay for DNA Damage Assessment

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The comet assay (single cell gel electrophoresis) was performed with the Trevigen Comet AssayTM kit (Trevigen) according to the manufacturer’s protocol. Briefly, IEC-6 cells were collected in PBS to a concentration of 1 × 105 cells/mL, mixed with 37 °C 1% LMA garose (low-melting agarose) and loaded on 2-well CometSlides. CometSlides were placed in the pre-cold lysis solution at 4 °C for 60 min, and then incubated in alkaline unwinding solution at room temperature for 20 min in the dark. CometSlides were transferred to a pre-cold fresh alkaline electrophoresis solution and subjected to electrophoresis using the CometAssay Electrophoresis System II (Trevigen) for 30 min (21 V). Slides were washed twice in dH2O for 5 min and 70% ethanol for 5 min. DNA was stained with 50 μL DAPI in a light-protected setting for 30 min and visualized using the confocal laser scanning microscope (CLSM, Carl Zeiss LSM 700).
Li M., Zhang R., Xin M., Xu Y., Liu S., Yu B., Zhang B, & Liu J. (2022). Discovery and Validation of Potential Serum Biomarkers with Pro-Inflammatory and DNA Damage Activities in Ulcerative Colitis: A Comprehensive Untargeted Metabolomic Study. Metabolites, 12(10), 997.
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