Comet assay was applied to quantify sperm DNA damage after thawing. This technique was performed by applying the method described for C. angulata sperm by Riesco et al. (2019) (link). Spermatozoa were embedded in low melting point agarose (0.5% w/v) on agarose precoated slides. Slides were immersed in lysis solution (2.5 M sodium chloride (NaCl), 100 mM ethylenediaminetetraacetic acid disodium salt dihydrate, 10 mM Tris, 1% Triton X-100, 1% lauryl sarcosine, pH 10, 1 h at 4°C). Then, slides were immersed twice in neutralizing solution (0.4 M Tris, pH 7.5, 5 min at 4°C) and fixed in 100% ethanol (3 min). Slides were stained with propidium iodide (PI) (0.1 mg/ml) and photographed with a digital camera (F-view, Olympus Corporation, Tokyo, Japan) coupled to a fluorescent microscope (excitation filter 450–480 nm; Olympus IX 81, Olympus Corporation, Tokyo, Japan). Komet 6.0 software (Andor Technology Ltd., Belfast, United Kingdom) was used to quantify the percentage of DNA in tail of 100 cells per slide. Two slides were performed per sample (n = 5).
Komet 6
Manufactured by Oxford Instruments
Sourced in United Kingdom
Komet 6.0 is a software product developed by Oxford Instruments. It is designed to analyze and process data from various analytical instruments and techniques. The software provides tools for data acquisition, visualization, and analysis, but its core function is to enable users to interpret and understand the data generated by their laboratory equipment.
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10 protocols using komet 6
1
Comet Assay for Sperm DNA Damage
2
Alkaline Comet Assay for DNA Damage
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Alkaline comet assay was performed as described (Magalhães et al., 2018 (link)). Briefly, cells were exposed to UV and collected at different timepoints. Cells were mixed with 0.5% low melting-point agarose and applied onto a glass slide covered with a thin layer of 1.5% agarose. Then cells were lysed and submitted to electrophoresis at constant voltage of 25 V for 30 min. The slides were neutralized with 0.4 M Tris-HCl pH 7.5, fixed with ethanol and stained with 2 μg/mL ethidium bromide. 100 nuclei from each slide were photographed in a fluorescence microscope (Olympus BX51). DNA fragmentation was expressed as the Olive Tail Moment (OTM) parameter by using the Komet 6.0 software (Andor Technology). Data in form of bars graph were also submitted to a linear regression analyses using the post-irradiation time-points to estimate the repair rate, where the repair speed was considered proportional to the slope.
3
UVR-Induced DNA Damage Assay in MeWo Cells
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Parental MeWo cells and MeWo-RhoA-N19 and MeWo-RhoA-V14 mutant cells were plated at a density of 2 × 105 cells/plate on 35 mm plates 24 h before UVA, UVB, or UVC irradiation. Following each specific treatment, the cells were collected via trypsinization and mixed with 0.5% low-melt agarose at 37°C. This mixture was applied to glass slides covered with a thin layer of 1.5% agarose and incubated at 4°C for 15 min for jellification. The cells were subsequently lysed in lysis solution (10 mM Tris, pH 10; 2.5 M NaCl; 100 mM EDTA; 1% Triton X-100; and 10% DMSO) for 24 h at 4°C. Following lysis, the slides were placed in a horizontal electrophoresis tank, immersed in electrophoresis buffer (300 mM NaOH and 1 mM EDTA), and incubated for 30 min to denature the DNA. The slides were subjected to electrophoresis at 1 V/cm and 300 mA for 30 min. Subsequently, the slides were incubated in neutralization buffer (0.4 M Tris-HCl, pH 7.5) for 15 min and fixed in absolute ethanol for 5 min, followed by DNA staining with 2 μg/mL ethidium bromide and visualization under a fluorescence microscope (Olympus BX51). The results of the DNA damage analysis assay were expressed as the olive tail moment, which was obtained using Komet 6.0 software (Andor Technology, Oxford, UK), and 100 cells per sample were analyzed (50 cells per slide) [28 (link)].
4
Comet Assay for DNA Damage
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Twenty-four hours post transfection, nearly 3 × 104 cells (in 1 X DPBS) were added to 0.75% low melting agarose. Single cell suspension was loaded onto glass slides (pre-coated with 0.1% low melting agarose) and allowed to cool at 4 °C. The cells were lysed by dipping slides into alkaline lysis buffer at 4 °C and washed in MilliQ. The slides were incubated in freshly prepared alkaline electrophoresis buffer at 4 °C and then run at 2 V/cm voltage and 300 mA for 20 mins. The excess alkali was neutralized with 0.4 M Tris and the slides were dried and 50 μM of propidium iodide was added to each slide just before imaging to stain the DNA. Images were obtained using a Leica fluorescence microscope at 40X. Both Olive Tail Moment and tail length were measured for 30 randomly selected comets per sample and analyzed using Komet 6.0 software (ANDOR technology, UK).
5
Comet Assay for DNA Interstrand Crosslinks
6
Quantification of DNA Interstrand Cross-Links
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In vitro DNA ICLs were quantified using the single-cell gel electrophoresis (comet) assay, as described previously (33, 35 (link)). Briefly, SN12C or MDA-MB-468 cell lines were incubated with ADCT-601, B12-PL1601, or the free PBD dimer cytotoxin SG3199 for 2 hours at 37°C; then cells were washed, resuspended, and incubated at 37°C for 36 hours. ADCT-601, B12-PL1601 or SG3199 treated cells were irradiated (15 Gy) and kept on ice. Cells were stained with propidium iodide and visualized under epifluorescence microscope staining; the resulting image resembles a “comet” with a distinct head and tail. The Olive tail moment (OTM), defined as the product of tail length and fraction of total DNA in the tail, was evaluated by Komet 6 software (Andor Technology). Percentage reduction in OTM observed in cells treated with ADCT-601, B12-PL1601 or SG3199 was calculated and compared with untreated cells to indirectly quantify the level of cross-linked DNA.
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In vitro DNA ICLs were quantified using the single-cell gel electrophoresis (comet) assay, as described previously (33, 35 (link)). Briefly, SN12C or MDA-MB-468 cell lines were incubated with ADCT-601, B12-PL1601, or the free PBD dimer cytotoxin SG3199 for 2 hours at 37°C; then cells were washed, resuspended, and incubated at 37°C for 36 hours. ADCT-601, B12-PL1601 or SG3199 treated cells were irradiated (15 Gy) and kept on ice. Cells were stained with propidium iodide and visualized under epifluorescence microscope staining; the resulting image resembles a “comet” with a distinct head and tail. The Olive tail moment (OTM), defined as the product of tail length and fraction of total DNA in the tail, was evaluated by Komet 6 software (Andor Technology). Percentage reduction in OTM observed in cells treated with ADCT-601, B12-PL1601 or SG3199 was calculated and compared with untreated cells to indirectly quantify the level of cross-linked DNA.
7
Alkaline Comet Assay for DNA Damage
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The alkaline comet assay was performed as described by Singh et al., 1998 [12 (link)], with modifications. HeLa cells were seeded in 35-mm dishes (2 × 105 cells/dish) and were allowed to adhere at 37°C (with 5% CO2) for 24 h, before γ-radiation with 5 Gy. After treatment, cells were harvested by trypsinization, mixed with 0.5% low-melting point agarose, and 100 μL of this mixture was pipetted onto glass slides with 1.5% normal-melting point agarose. Then, cells were lysed with lysis buffer (10 mmol/L Tris, pH 10, containing 2.5 mmol/L NaCl, 100 mmol/L EDTA, 1% Triton X-100, and 10% DMSO, all from Sigma-Aldrich, Saint Louis, MO, USA) for 24 h at 4°C and in the dark. Samples were denatured in alkaline electrophoresis buffer (300 mmol/L NaOH, 1 mmol/L EDTA, pH >13) for 25 min, and then electrophoresis was performed at 25 V and 300 mA, for 30 min. After electrophoresis, slides were washed 3 times (5 min/wash) in neutralizing buffer (0.4 mmol/L Tris-HCl, pH 7.5), DNA was stained with 2 μg/mL ethidium bromide, and comets (from 50 cells/slide, in duplicate) were imaged using a fluorescence microscope Olympus IX51 (Olympus, Shinjuku, Tokyo, Japan). Comet assay data were analyzed using the software Komet 6.0 (Andor, Technology, Belfast, BT, UK).
8
Comet Assay with Fpg Treatment
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The cells were seeded in 12-well plates at densities of 2 × 10 5 /well or 3 × 10 5 /well for a 24 or 72 h treatment, respectively. Hydrogen peroxide (H 2 O 2 ) at a concentration of 200 µmol was exposed to the cells as positive control for an hour. For each sample, two wells were prepared for both the conventional treatment and the formamidopyrimidine glycosylase (Fpg) treatment. Conventional comet assay was performed in alkaline conditions (pH > 13) as described previously [21] (Wang et al., 2018) [21] (Wang et al., 2018) [21] . For the Fpg treated wells, an additional Fpg treatment was applied before the DNA unwinding procedure, and the slides were immersed in an enzyme buffer (0.1 M KCl, 0.5 mM EDTA, 40 mM HEPES, 0.2 mg.mL - 1 BSA) three times for 5 min each. The Fpg (New England Biolabs, Inc. UK) was diluted at 1:50000 with enzyme buffer. 100 mL aliquots of the diluted enzyme were added to each gel on the microscope slides and incubated in a humidity chamber at 37 °C for 30 minutes. The remaining steps were the same as the conventional treatment. The comet assays were performed in triplicate. At least 50 cells per sample were independently scored using the Nikon Eclipse 80i uorescent microscope (Nikon, Tokyo, Japan), while Komet 6.0 (Andor Technology, Belfast, UK) was used to analyze the medium value of percentage DNA in tail and Olive tail moment (OTM) of each sample.
9
Comet Assay for Embryonic DNA Damage
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DNA strand break measurements by the alkaline comet assay were performed on 24 hpf embryos. Pools of five dechorionated embryos per replicate were digested with 1 mg.mL -1 of phosphate-buffered saline 1X/collagenase IV from Clostridium histolyticum (PBS 1 X, 137 mM NaCl • KCl 2.7 mM • Na2HPO4 10 mM • KH2PO4 1.8 mM, pH 7.4, Sigma-Aldrich, Germany)
during 45 minutes at room temperature. Cell suspension was filtered through a 48 µm gauze in order to separate individual cells from tissue debris. Following a centrifugation for 10 minutes at 2300 rpm at room temperature, cells pellet was resuspended in 30 µl of PBS. Then, the comet assay was performed as described by Akcha et al. (2003) . For each cell sample, two slides were prepared. DNA was stained with 70 µL of GelRed™ solution (1/10000) for one hour at 4 °C in the dark. Slides were analyzed using a fluorescence microscope (Olympus BX60, 400x) coupled to a Luca-S EMCCD camera (Andor™ technology, Northern Ireland) and imaging analysis software (Komet 6.0, Andor™ Technology, Northern Ireland). Genotoxicity was assessed by measuring the DNA percentage in the comet tail for at least 50 nuclei per slide.
during 45 minutes at room temperature. Cell suspension was filtered through a 48 µm gauze in order to separate individual cells from tissue debris. Following a centrifugation for 10 minutes at 2300 rpm at room temperature, cells pellet was resuspended in 30 µl of PBS. Then, the comet assay was performed as described by Akcha et al. (2003) . For each cell sample, two slides were prepared. DNA was stained with 70 µL of GelRed™ solution (1/10000) for one hour at 4 °C in the dark. Slides were analyzed using a fluorescence microscope (Olympus BX60, 400x) coupled to a Luca-S EMCCD camera (Andor™ technology, Northern Ireland) and imaging analysis software (Komet 6.0, Andor™ Technology, Northern Ireland). Genotoxicity was assessed by measuring the DNA percentage in the comet tail for at least 50 nuclei per slide.
10
Comet Assay for DNA Strand Breaks in Zebrafish Embryos
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DNA strand break measurements by the alkaline comet assay were performed on 24 hpf embryos. Pools of five dechorionated embryos for four samples per concentration were digested with 1 mg.mL -1 of phosphate-buffered saline 1X/collagenase IV from Clostridium histolyticum (PBS 1X, 137 mM NaCl • KCl 2.7 mM • Na 2 HPO 4 10 mM • KH 2 PO 4 1.8 mM, pH 7.4, Sigma-Aldrich, Germany) solution during 45 minutes at room temperature. Cell suspension was filtered through 48 µm gauze into a 1.5 mL reaction tube in order to separate individual cells from tissue debris. Following a centrifugation for 10 minutes at 2300 rpm at room temperature, the cells pellet was resuspended with 30 µl of PBS. The viability of the dissociated embryo cells was evaluated by a Trypan blue exclusion test. Then, the comet assay was performed as described by Akcha et al. (2003) . Two slides were prepared per sample. DNA was stained with 70 µL of GelRed™ solution (1/10000) for one hour at 4 °C in the dark. Slides were analyzed using a fluorescence microscope (Olympus BX60, 400x) coupled to a Luca-S EMCCD camera (Andor™ technology, Northern Ireland) and imaging analysis software (Komet 6.0, Andor™ Technology, Northern Ireland). Genotoxic assessment was assessed by measuring the DNA percentage in the comet tail (50 cells per slide).
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