Role of Sperm DNA Damage in Male Infertility Assessment
Botros Rizk, Ashok Agarwal, Edmund S. Sabanegh in Male Infertility in Reproductive Medicine, 2019
The Comet assay measures the amount of DNA damage per spermatozoon [50]. The steps involved are embedding cells in agarose, lysis under neutral or alkaline conditions, subjecting the lysed cells to electrophoresis, DNA staining, and microscopic image analysis. The principle underlying the assay involves unwinding the tightly packed sperm nucleus and subjecting the spermatozoon to electrophoresis that enables the detection of the amount of fragmented DNA that migrates away from the sperm head. The damaged cell resembles a comet with the fragmented DNA in the comet’s tail and the intact DNA in the head. The staining intensity and length of the comet’s tail indicate the degree of SDF. In contrast to other DNA fragmentation assays, Comet assay provides qualitative and quantitative insight into DNA damage on a single cell, instead of providing a proportion of DNA-damaged cells [51]. Comet detects ss and ds breaks, as well as abasic sites. It has higher sensitivity and specificity than other DNA fragmentation tests (SCD, TUNEL, and SCSA). In addition, it requires only a small number of cells for the evaluation of DNA fragmentation; thus, it can be employed in cases of severe oligozoospermia [52].
Anticancer Properties of Silver Nanoparticles from Root Extract of Trigonella Foenum-Graecum
Megh R. Goyal, Preeti Birwal, Santosh K. Mishra in Phytochemicals and Medicinal Plants in Food Design, 2022
DNA fragmentation in the AgNPs treated cells and control cells were used to observe in the comet assay. Briefly, each sample three slides were prepared: (1) 1st layer contains with 400 µL of 1% LMPA at 37 °C; (2) second layer cells contains with 25 µL in each sample along 0.5% LMPA for 175 µL at 37 °C; (3) third layer contains with 200 µL of 1% LMPA at 37 °C. The coverslips were detached in each layer and the slides were exposed to lysis (at 4 °C for 1 h to protect from light) by existence flooded in cells lysis buffer containing with 2.5 M NaCl and 100 mM EDTA, 1% TritonTM X-100, 10 mM Tris pH at 10, and 10% DMSO, then slides calibrated for 20 min in electrophoresis buffer pH at 13 that containing 300 mM NaOH, 1 mM Na2EDTA, and finally electrophoresis (300 mA, 25V, 35 min). After electrophoresis, slides wash away three times for 5 min each wash in 0.4 M Tris-buffer (pH 7.4) then marked at 40 µL EtBr. After washing, coverslips laid on slides and kept at 4 °C overnight and observed by fluorescent microscope manufacturing by Olympus IXSI inverted microscope, images captured, and comet tails of 50 cells using Soft Imaging System (Life Science, analySISW v5). The obtained data remained stated as mean tail length in micrometer.
Structure-Function Elucidation of Flavonoids by Modern Technologies
Dilip Ghosh, Pulok K. Mukherjee in Natural Medicines, 2019
COMET assay is one of the standard methods for assessing DNA damage. Cook et al. (1976) first studied the nuclear structure based on the lysis of the cells using non-ionic detergent and sodium chloride. Based upon their study, Ostling and Johanson (1984) modified the technique and described the tails in terms of DNA supercoiling. However the term “COMET” is used due to pattern of migration of the DNA fragments through the electrophoresis gel (Tice et al. 2000). The basic principle of the assay is that the cells are first encapsulated in agarose and then lysed with detergents and high salts so that nucleoids having supercoiled DNA will be formed. The cells having damaged DNA will have many more short fragments in comparison to those having intact DNA. These cells, when electrophoresed, will have a larger tail-like stretch than normal cells. This is because the nucleoids, having breaks, will lose their supercoiling and tends to move towards anode. Later any dye intercalating or binding DNA can be used to detect the pattern of migration, which in turn can be visualized in a florescent microscope. The DNA damage is then quantified based upon the migration scores either performed manually or by software (Klaude et al. 1996; Collins et al. 1997). Currently there are different variants of the COMET assays. Some important variants include alkaline single cell gel electrophoresis (Singh et al. 1988; Olive et al. 1990), neutral single cell gel electrophoresis (Collins 1999), fluorescent in situ hybridization comet assay (Santos et al. 1997; Rapp et al. 1999; Mladinic et al. 2012) and bromodeoxyuridine labelled detection (McGlynn et al. 1999). Tice et al. (2000) provided a detailed protocol for the COMET assay.
Characterization and cytotoxicity assessment of nargile smoke using dynamic exposure
Published in Inhalation Toxicology, 2019
Christian Khalil, Joe Braham Chahine, Brenda Chahla, Tamara Hobeika, Rony S. Khnayzer
The comet assay is a technique to measure DNA damage in exposed cells as previously described (Soni et al. 2010; Khalil 2015; Khalil and Shebaby 2017). The kit provided by Trevigen was utilized according to manufacturer instructions. In summary, the agarose provided in the kit was incubated at 37 °C in a water bath prior to use. The cells were counted and a density of 105 cells/ml was added to the molten agarose (1:10 v/v). This was followed by pipetting the mixtures to the comet slides provided. The slides where then stored in the fridge for 30 min to allow the agarose to solidify. Upon agarose, solidification slides were immersed in pre-chilled lysis solution for one hour. (Al Hageh et al. 2018). This was followed by alkaline phosphatase treatment, followed by Tris base, boric acid and EDTA (TBE) buffer immersion prior to electrophoresis at 1 V/cm for 10 min. Upon electrophoresis; slides were subjected to ethanol treatment then dried prior to silver staining for comet visualization. The silver stained slides were further analyzed using Comet Analysis Software Package. The generated parameter data by the software was used to calculate the Tail Moment Index (TMI). The TMI calculation consisted of the average of tail DNA values multiplied by average tail length divided by 1000.
Comet assay for assessment of DNA damage in greenhouse workers exposed to pesticides
Published in Biomarkers, 2019
Akin Cayir, Mahmut Coskun, Munevver Coskun, Hayal Cobanoglu
A biomarker is a cellular, biochemical or molecular alteration that can be easily and non-invasively measured in human tissues. It directly or indirectly provides information about environmental exposure to hazardous agents and also indicate risk of future disease (Collins et al.2014). Measurement by means of biomarkers in a human population exposed to mutagens or carcinogens is accepted as an early detection system for exposure. The comet assay is a well-established, simple, versatile, rapid, visual, sensitive and extensively used tool to assess DNA damage and repair, quantitatively as well as qualitatively, in individual cell populations (Dhawan and Anderson 2009). The assay has been used to test the genotoxic potential of a broad range of chemicals (Guilherme et al.2012, Bokán et al.2013, de Barros et al.2013, Syberg et al.2015) and to determine the potential DNA damage level on the human population (Costa et al.2011, Kaur et al.2011, Benedetti et al.2013). When compared with similar methods, the comet assay is a less time-consuming and laborious experimental procedure.
Gentiopicroside Inhibits Cell Growth and Migration on Cervical Cancer via the Reciprocal MAPK/Akt Signaling Pathways
Published in Nutrition and Cancer, 2021
Zhiqiang Hu, Hui Wang, Yan Fu, Kang Ma, Xiaoyan Ma, Jing Wang
For further assessment, the nuclear morphological change of apoptotic cell, Hoechst 33342 staining, and comet assay was conducted. After HeLa cells stained with Hoechst 33342, an increased number of cells were observed with bright nuclear condensation and fragmentation of nuclei, which were regarded as characteristics of cell apoptosis (Fig. 3B-a). Comet assay is a simple and fast method to detect DNA damage, which is widely used in the fields of cell apoptosis. By measuring the tail length and tail DNA, DNA damage could be quantitatively analyzed. As shown in Fig. 3B-b, GPS caused comet formation in HeLa with the addition of concentration. Furthermore, the tail area, tail DNA, tail DNA percentage, tail length, and tail moment were all significantly increased (Table 1), which further suggested cell apoptosis after GPS treatment (P < 0.01 or P < 0.001).
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