Assessments of DNA double strand break repair by PCR after I-PpoI induction were performed as previously described (23 (link)). Comet assays were performed using the Trevigen CometAssay Kit (Trevigen, Gaithersburg, MD) according to the manufacturer's protocol. Cell images were assessed by the TriTek CometScore 1.6.1.21 software. Olive tail moment (OTM) was determined as a product of DNA in the tail and the mean distance of migration in the tail.
Trevigen comet assay kit
Manufactured by Bio-Techne
Sourced in United States
The Trevigen Comet Assay kit is a laboratory tool used to measure DNA damage and repair at the cellular level. The kit provides reagents and protocols to perform the comet assay, a single-cell gel electrophoresis technique that allows visualization and quantification of DNA fragmentation within individual cells.
Automatically generated - may contain errors
Lab products found in correlation
Sybr gold, by Bio-Techne (3 mentions)
Bx53 fluorescence microscope, by Olympus (2 mentions)
Eclipse 80i, by Nikon (2 mentions)
Fluorescence microscope, by Nikon (2 mentions)
Cometslide, by Bio-Techne (1 mentions)
Dp72 camera, by Olympus (1 mentions)
N tetraacetic acid, by Merck Group (1 mentions)
Sodium hydroxide (naoh), by Nacalai Tesque (1 mentions)
Sybr green 1, by Olympus (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Lysis solution, by Bio-Techne (1 mentions)
Sybr green 1, by Bio-Techne (1 mentions)
Eclipse ni u, by Nikon (1 mentions)
Lsm 800 laser scanning microscope, by Zeiss (1 mentions)
Comet assay 4 program, by Oxford Instruments (1 mentions)
Axiocam mrm camera, by Zeiss (1 mentions)
Axiovert 200, by Zeiss (1 mentions)
Sybr green, by Thermo Fisher Scientific (1 mentions)
Sybr green dye, by Bio-Techne (1 mentions)
Eclipse ti microscope, by Nikon (1 mentions)
29 protocols using trevigen comet assay kit
1
DNA Double Strand Break Repair Analysis
2
Alkaline Comet Assay for Genotoxicity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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)).
3
Comet Assay for DNA Strand Break Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The comet assay was performed using a Trevigen Comet Assay Kit (Trevigen Inc., Gaithersburg, MD, USA) to detect DNA strand breaks at the single-cell level as described previously [18 (link)]. Briefly, cells were exposed to millimeter-wavelength radiation for 24 h, collected by trypsinization and centrifuged immediately after exposure, then mixed with low melting point agarose to prepare a cell suspension in 0.1% agarose/phosphate buffered saline (PBS). After gelation of the agarose, the cells were lysed, then the alkaline unwinding was performed (1 h at 4 °C, pH > 13). The resulting DNA samples were electrophoresed at 1 V/cm for 30 min in a 0.3 M NaOH (Nacalai Tesque, Kyoto, Japan) and 1 mM ethylenediamine-N,N,N’,N’-tetraacetic acid (Sigma-Aldrich) solution. After the DNA was stained with SYBR Green I, immunofluorescence images were captured using a fluorescence microscope (Olympus, Tokyo, Japan). DNA strand breaks were analyzed using Comet software (Perceptive Instruments, Suffolk, UK). At least 100 comets from each gel were analyzed, and at least five independent experiments were performed. Tail length indicates the pixel length of the comet tail, tail percent indicates the percentage of tail content relative to comet content, and tail moment was calculated as follows:
4
Comet Assay for DNA Damage Assessment
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Comet assay was performed by using the Trevigen comet assay kit (Trevigen, 4250-050-K) following the manufacturer’s instruction. Briefly, nuclei extracted from 22-day-old rosette leaves at 1 x 105/mL were combined with molten LMAgarose at a ratio of 1:10 (v/v) at 37°C. Samples (50 μL) were immediately pipetted onto CometSlide. The slides were placed flat at 4°C in the dark for 30 min and then immersed in prechilled Lysis Solution (Trevigen, 4250-050-01,) at 4°C for 60 min. After lysis, the slides were immersed in freshly prepared Alkaline Unwinding Solution, pH >13 (200 mM NaOH, 1 mM EDTA) for 20–60 min at room temperature in the dark. Electrophoresis was in Alkaline Electrophoresis Solution, pH >13 (200 mM NaOH, 1 mM EDTA) at 21 volts for 30 min at 4°C. After washing in dH2O and 70% ethanol, samples were dried at ≤ 45°C for 10–15 min. DNA was stained by diluted SYBR Green I (Trevigen, 4250-050-05,) in refrigerator for 5 min. The slides were dried completely at room temperature in the dark and then viewed by Olympus BX53 fluorescence microscope. The Comet Score software (http://www.autocomet.com ) was used to evaluate the levels of DNA damage.
5
Alkaline Comet Assay for DNA Damage
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Alkaline comet assay was performed using Trevigen CometAssay® Kit (Trevigen, Gaithersburg, MD, USA). Detached cells were resuspended in cold PBS to a concentration of 105 cells/mL. An aliquot of 10 μL of cell suspension was added to 100 μL of melted LM agarose maintained at 37°C. The agarose mixture was transferred onto a comet slide and allowed to solidify at 4oC. Then the cells were lysed in pre-chilled lysis solution for 60 min at 4°C, and denatured at room temperature in an alkaline solution containing 300 mM NaOH and 1 mM EDTA for 60 min. Electrophoresis was carried out under 1 V/cm condition in a solution (300 mM NaOH and 1 mM EDTA) for 30 min. All incubations and electrophoresis were performed in dark. The slides were stained with SYBR Green for 20 min and immediately photographed using a fluorescence microscope (Nikon Eclipse Ni-U). Comet tail was analyzed using the Comet Assay Software Project tool [57 (link)].
6
Alkaline Comet Assay for DNA Damage
7
Alkaline Comet Assay for DNA Damage Analysis
8
DNA Damage Quantification by Comet Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The neutral and alkaline comet assay were performed on the Trevigen Comet Assay kit (Cat. 4252-040-K, Trevigen, Gaithersburg, Maryland, USA). Cells were pretreated with 0.5 mM VPA for 24 h followed by 8 Gy IR treatment. Low melting agar was combined with cell suspension at a ratio of 1:10 after treatments, then spread onto comet slide immediately. The slides were cooled for 30 min, then immersed in lysis solution. Slides were immersed into cold alkaline unwinding solution for 1 h (alkaline comet), then transferred into neutral or alkaline electrophoresis buffer at 21 V for 30 min at 4°C in the dark. The slides were washed twice in distilled water and 70% ethanol for 5 min, then dried and stained with SYBR gold (1:10000, Trevigen).
9
Comet Assay to Assess DNA Damage
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Jab1-specific or control siRNAs were transfected into SNU-478 and HuCCT-1 cell lines daily for 2 days. These cell lines were treated with 2.5 μM or 5 μM cisplatin for 48 hours. The cells were then trypsinized and subjected to an alkaline comet assay using the Trevigen Comet Assay Kit (Trevigen Inc., Gaithersburg, MD) following the manufacturer’s protocol. Tail moments and tail intensity were measured with Comet Assay IV software (Perceptive Instruments Ltd., Bury St. Edmunds, UK). Three independent experiments were performed for each condition.
10
Quantifying DNA Damage Using Comet Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were seeded at a density of 2×105 cells/well in a 6 cm well dish. After treatment with cisplatin or M3814 or with a combination of cisplatin and M3814 for 72 hours, cells were trypsinized, and DNA damage was analyzed using the Trevigen CometAssay kit (Trevigen, Gaithersburg, Maryland, USA). The alkaline comet assay was used to assess DNA damage in all cells, including PRKDC-knockdown cells and drug-treated cells, according to manufacturer’s instructions. Cells that were embedded on a glass slide in agarose were stained with SafeView nucleic acid stains (Applied Biological Materials, Richmond, Canada). Cells were observed using a fluorescence microscope for quantification; comets were analyzed as 100 cells per replica, and the DNA tail intensity was calculated using OpenComet, a plug-in processing platform in ImageJ (National Institutes of Health, USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!