Alkaline comet assays were performed with manufacturer’s instruction (Trevigen). Cells were trypsinized and suspended in cold PBS at a concentration of 2.0 × 105 cells/ml. The cells were then mixed with low melting agarose (Trevigen) at a ratio of 1:10 (v/v) and immediately plated onto Cometslide (Trevigen). Alkaline electrophoresis was run at 25 V for 30 minutes in the electrophoresis system. Data were analyzed with a fluorescence microscope (Zeiss).
Low melting agarose
Manufactured by Bio-Techne
Sourced in United States
Low melting agarose is a type of agarose gel used in various laboratory applications. It has a lower melting and gelling temperature compared to standard agarose, allowing for gentle handling and manipulation of biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Cometslide, by Bio-Techne (5 mentions)
Sybr green, by Bio-Techne (1 mentions)
Cell f software, by Olympus (1 mentions)
Sybr green 1, by Thermo Fisher Scientific (1 mentions)
Gelbond film, by Lonza (1 mentions)
Ix71 fluorescent microscope, by Olympus (1 mentions)
Fluoview fv1000 confocal microscope, by Olympus (1 mentions)
Lysis solution, by Bio-Techne (1 mentions)
Sybr gold, by Qiagen (1 mentions)
7 protocols using low melting agarose
1
Alkaline Comet Assay Protocol
2
Detecting ssDNA and dsDNA Damage
3
Comet Assay Reveals Allopurinol-AZA Toxicity
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The comet assay was used to identify the mechanisms behind the increased toxicity with allopurinol co-administration. This assay was performed according to the manufacturer’s instructions (Trevigen Inc., Gaithersburg, MD). HepaRG cells were seeded in 25-cm2 flasks and treated 24 h with allopurinol (100 μM), AZA (70 μM), AZA (70 μM) + allopurinol (100 μM) or untreated (control). After 24 h, cells were harvested and the cell suspension was mixed with 75 μL low melting agarose (Trevigen Inc.) in a density of 2 × 104 cells/mL and directly pipetted on agarose-precoated slides. Slides were stored at 4 °C for 30 min and subsequently submerged in lysis solution. After 60-min lysis, they were treated with alkaline unwending solution (pH > 13) for 60 min, followed by 30-min electrophoresis at 25 V. Slides were stained with SYBR green (Trevigen Inc.) and visualised and photographed by a digital camera (AxioCam MRm) attached to a fluorescent microscope (Axio imager.M1) using a 20× magnification.
4
Neutral Comet Assay for DSB Detection
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Neutral comet assay can mainly detect double‐strand breaks (DSBs) at the individual cell level.[51 (link)
] In brief, cells were trypsinized and suspended in cold 1×PBS (Ca2+ and Mg2+ free), mixed with low melting agarose (Trevigen) at a ratio of 1:10 (v/v), and immediately plated onto Cometslide (Trevigen). Neutral electrophoresis was run at 25 V for 30 min in the electrophoresis system. Cell comets were imaged using a fluorescence microscope.
] In brief, cells were trypsinized and suspended in cold 1×PBS (Ca2+ and Mg2+ free), mixed with low melting agarose (Trevigen) at a ratio of 1:10 (v/v), and immediately plated onto Cometslide (Trevigen). Neutral electrophoresis was run at 25 V for 30 min in the electrophoresis system. Cell comets were imaged using a fluorescence microscope.
5
Quantifying DNA Damage by Comet Assay
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U2OS cells were treated with G9a inhibitor (UNC0638, 1 µM) or ATM inhibitor (KU55933 [16] (link), 10 µM) for 24 h, while HCT116 p53 WT/KO cells were treated for 4 days. For siRNA-transfected cells, assays were performed 72 h after transfection. Neutral comet assay were performed with the damaging agent phleomycin, as previously reported [17] (link). Briefly, an appropriate number of cells were mixed with low-melting Agarose (Trevigen) and bound on GelBond film (Lonza). Samples were lysed and electrophoresed at 35 V for 7 min. The samples were fixed, dried and stained with SYBR Green I (Invitrogen). Images were taken with an IX71 fluorescent microscope (Olympus) using Cell^F software (Olympus). Tail moments were quantified using CometScore software (TriTek). Means of tail moments of at least 50 cells were measured per condition.
6
Alkaline Comet Assay for DNA Damage
7
Comet Assay for DNA Damage Evaluation
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First, 400,000 cells per well were seeded on a 12-well plate overnight, treated accordingly, harvested, and resuspended in 100 µL PBS. Then, 20 µL of this suspension was mixed with low-melting agarose (Trevigen, Gaithersburg, MD, USA), spread evenly over CometSlides (Trevigen, Gaithersburg, MD, USA), left to congeal, and then kept in lysis solution (Trevigen) at 4 °C overnight. Thereafter, the slides were immersed in unwinding solution for 30 min at room temperature before gel electrophoresis was run for 25 min. The slides were washed, dried at 37 °C overnight, and stained with SYBRGold (Qiagen, Hilden, Germany). Fluorescence images were taken with Olympus Fluoview FV1000 confocal microscope (Olympus, Tokyo, Japan) and analysed using OpenComet [93 (link)]. The tail moment and the olive moment were calculated as follows:
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