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.
Cometslide
Manufactured by Bio-Techne
Sourced in United States
CometSlides are a type of laboratory equipment designed for performing the comet assay, a widely used technique in the field of DNA damage and repair analysis. The core function of CometSlides is to provide a standardized platform for the preparation and analysis of cells or tissues subjected to the comet assay.
Automatically generated - may contain errors
Lab products found in correlation
Sybr gold, by Thermo Fisher Scientific (17 mentions)
Lysis solution, by Bio-Techne (13 mentions)
Comet assay kit, by Bio-Techne (12 mentions)
Lmagarose, by Bio-Techne (10 mentions)
Sybr green, by Thermo Fisher Scientific (7 mentions)
Low melting agarose, by Bio-Techne (5 mentions)
Comet lm agarose, by Bio-Techne (5 mentions)
Sybr gold nucleic acid gel stain, by Thermo Fisher Scientific (4 mentions)
Lysis buffer, by Bio-Techne (4 mentions)
Lsm 710, by Zeiss (4 mentions)
Cometassay lysis solution, by Bio-Techne (3 mentions)
Cygreen, by Enzo Life Sciences (3 mentions)
Cometassay, by Bio-Techne (3 mentions)
Sybr green 1, by Bio-Techne (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Cometassay electrophoresis system, by Bio-Techne (3 mentions)
Sybr green, by Bio-Techne (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Dfc345 fx camera, by Leica (2 mentions)
Dmu 4000b, by Leica (2 mentions)
112 protocols using cometslide
1
Comet Assay for Cellular DNA Damage
2
Comet Assay Protocol for DNA Damage
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A comet assay was performed using a CometAssay Kit (Trevigen, Gaithersburg, MD, USA) according to the manufacturer’s protocol. Briefly, GS cells were cultured for 2 weeks after adenovirus infection and suspended in PBS (2 × 105 cells/ml). The cell suspension was mixed with CometAaay LM Agarose (Trevigen) at a ratio of 1:10, and 50 μl was transferred to a CometSlide (Trevigen) for immobilization. After incubation in lysis solution, cells were treated with an alkaline solution for 30 min followed by electrophoresis. DNA was stained with SYBR Green I (TaKaRa Bio, Otsu, Japan). DNA damage was quantified by measuring the length of the visible comet tail. Cells with a ratio greater than or equal to 2 were counted as positive for DNA damage. Figures show the percentage of cells positive for DNA damage per treatment.
3
Alkaline Comet Assay for Genotoxicity
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The alkaline comet assay was performed using the Trevigen CometAssay® Kit (Trevigen, Inc.; Bio-Techne) according to the manufacturer's instructions. Briefly, HepG2 cells were seeded at 8×104 cells/well in a 12-well plate. After 24 h, the cells were treated with vehicle control, 5 µΜ DEX, 1 µΜ EPI or 1 µΜ NE for 4 h in a 5% CO2 incubator at 37°C. Then the cells were harvested, washed twice with phosphate-buffered saline (PBS) and mixed with 1% low-melting agarose in PBS at 37°C to form a single cell suspension embedded in agarose. The resulting mixture was immediately pipetted on with the sample area of a CometSlide (Trevigen, Inc.; Bio-Techne). The slides were placed at 4°C for 10 min and then lysed at 4°C overnight in the dark. After lysis, the slides were subjected to electrophoresis at 21 V for 45 min at 4°C, and then immersed twice in distilled water for 5 min and once in 70% (v/v) ethanol for 5 min. The slides were dried completely at 37°C for 10–15 min and then stained with propidium iodide (PI) for 10 min at room temperature. Comets were observed using an Olympus BX53 fluorescence microscope (Olympus Corp.) equipped with an Olympus DP72 camera (Olympus Corp.). The percentage of DNA in the tail was quantified based on ≥30 randomly selected cells in each sample using Image J software (version 1.53c; National Institutes of Health) as previously described (37 (link)).
4
Immunocytochemistry and Comet Assay Protocol
5
Comet and FLARE Assays for DNA Damage
6
Neutral Comet Assay for DNA Damage
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Neutral comet assay was performed according to the Trevigen Comet Assay protocol (105 (link)). Cells were collected and resuspended in 1% low-melting agarose (Trevigen, 4250-50-050-02), spread onto a comet slide (Trevigen, 4250-200-03), and allowed to dry. Cells were then lysed in lysis solution (Trevigen, 4250-050-01) at 4°C for 1 hour. Slides were immersed in TBE buffer (0.1 M tris base, 0.1 M boric acid, and 2.5 mM EDTA) for 30 min before electrophoresis at 25 V for 30 min at 4°C. DNA was precipitated with 1 M ammonium acetate in 95% ethanol for 30 min and subsequently fixed in 70% ethanol for 30 min. Comets were stained with SYBR Gold (Thermo Fisher Scientific) for 30 min. Images were acquired with a Leica DM4B microscope with 10× objective. At least 150 comets were scored for each sample using the OpenComet plugin in the ImageJ analysis software (RRID: SCR_003070). Olive moment values are represented as Tuckey boxes.
7
Alkaline Comet Assay Protocol
8
Neutral Comet Assay for DNA Damage
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Neutral comet assay was performed as previously described (40 (link)). Briefly, 700 cells were resuspended in 70 μl 0.5% low melting point agarose (Trevigen, Cat. No. 4250-050-02) and spread on a comet slide (Trevigen, Cat. No. 4250-200-03). Cells were lysed in a cold lysis solution (Trevigen, Cat. No. 4250-050-01) at 4°C for 30 min. DNA migration was performed in TBE buffer at 1 V/cm for 30 min. Slides were washed in milliQ water, fixed with ethanol 70% for 30 min and dried at room temperature. Comets were labeled with SYBR® Gold Nucleic Acid Gel Stain (ThermoFisher) for 30 min. Images were acquired with a fluorescence microscope (LEICA DMU 4000B; 20×/0.4 CORR) coupled to the LEICA DFC345FX camera. The images were analyzed using the OpenComet plug-in from ImageJ. At least 100 comets were scored per sample in each experiment. Olive moment measures DSBs as a product of the amount of DNA present (intensity) times the size of the broken DNA pieces (length) in the tail.
9
Neutral Comet Assay for DNA Double-Strand Breaks
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A neutral comet assay was used to specifically assess the amount of DNA DSBs in cells treated with Veliparib, CPT, or both. In individual wells of a six-well plate, cells were incubated with 0.4 mL of Trypsin for 10 minutes. 1 mL of medium was added to the trypsin and the cells centrifuged at 1000 g for 5 minutes. The supernatant was removed, and the cell pellet was resuspended in 500 μL of PBS. The suspended cells were added to prewarmed low-melting agarose at 37°C (10 μL of cells to 90 μL of agarose). The mix was pipetted onto a CometSlide (4250, Trevigen) and spread equally across the slide before being allowed to set for 30 minutes at 4°C. Following this, the slides were submerged in cold Lysis Buffer (4250, Trevigen) for 30 minutes on a shaker, and then in cold Tris/Borate/EDTA buffer. Electrophoresis was run for 15 minutes at 21V in a Mini-Sub Cell GT electrophoresis tray (Bio-Rad). Subsequently, cells were fixed in 70% ethanol and allowed to dry overnight. DNA was stained with Cygreen (GEN-105, ENZO) for 30 minutes, and slides were imaged on an EVOS fluorescence microscope (AMG) with appropriate filters for GFP imaging. Images acquired were processed using Open Comet software and the olive moment for each group of cells was calculated [83 (link)].
10
Neutral Comet Assay for Apoptosis
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