Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells

Östling, O.; Johanson, Karl J.

doi:10.1016/0006-291x(84)90411-x

Cited by 1,412 publications

(641 citation statements)

References 7 publications

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“…Since introduction of the SCGE protocol (Ostling and Johanson, 1984), there have been modifications of it for use with various mammalian cell types, including sperm (Fairbairn et al, 1995). To test the SCGE technique with equine sperm, we induced damage by freezing, then thawing for 1, 3, and 5 cycles in a phosphate-buffered saline medium that was not suitable for cryopreservation.…”

Section: Discussionmentioning

confidence: 99%

“…Single-cell gel electrophoresis (SCGE) was first introduced in 1984 (Ostling and Johanson, 1984) and has become a recognized method for detecting DNA damage in a variety of vertebrate cell types, including sperm. The SCGE technique has been used to examine human sperm DNA integrity as it relates to semen quality (Hughes et al, 1999;Irvine et al, 2000), compare the damage induced by oxidation and x-irradiation to infertility (Hughes et al, 1999;Shen and Ong, 2000), or to investigate potential damage as a result of common semen handling practices, such as cryopreservation (Steele et al, 2000).…”

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confidence: 99%

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Detection of DNA Damage in Response to Cooling Injury in Equine Spermatozoa Using Single‐Cell Gel Electrophoresis

Linfor

Meyers

2002

Journal of Andrology

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Single-cell gel electrophoresis (SCGE), or comet assay, has the ability to detect damage at the single cell level and has not been reported for equine sperm. The ability to detect nuclear damage at the single cell level could aid in the advancement of protocols for optimal semen preservation. The goals of these experiments were to adapt this assay for use with equine sperm and to utilize the assay for determining the integrity of equine sperm DNA following treatments with storage at various decreased temperatures (Ϫ20ЊC and 5ЊC). Results from experiments in which sperm were frozen (Ϫ20ЊC) in the absence of cryoprotectants revealed that significantly more cells with fragmented tails of DNA, or comets, occurred among those exposed to 1, 3, and 5 freeze-thaw cycles (65% Ϯ 6%, 76% Ϯ 11%, 92% Ϯ 6%, respectively) compared with fresh, untreated sperm (19% Ϯ 16%, P Ͻ .05). In addition, DNA damage was different (P Ͻ .05) between the three freeze-thaw treatments. Sensitivity of SCGE on equine sperm was further tested with known ratios of frozen-thawed and fresh cells. The amount of detectable DNA damage was positively correlated with the percentage of cryodamaged cells in each treatment (r 2 ϭ 0.92, P Ͻ .05). Potential damage as a result of cooled storage was also investigated and results revealed that sperm stored for 48 hours (at 5ЊC) had a higher percentage of comets than that of fresh sperm (63% Ϯ 13.9% and 28% Ϯ 15.6%, respectively, P Ͻ .05). The percentage of viable sperm also decreased linearly over time and was inversely correlated with percent of comets (r 2 ϭ 0.805, P Ͻ .001). Detection of sublethal and/or uncompensable fertility factors in semen, such as DNA fragmentation, could be useful for detecting male differences in semen for cooling or cryopreservation potential and could provide a tool for monitoring and preserving fertility for individual stallions.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Detection of DNA Damage in Response to Cooling Injury in Equine Spermatozoa Using Single‐Cell Gel Electrophoresis

Linfor

Meyers

2002

Journal of Andrology

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“…(Here, we use the term "myocyte" to refer to all muscle-derived cells, including muscle satellite cells and differentiated myotubes). We then assessed the capacity of myocytes to repair DNA damage using the comet assay (28)(29)(30)(31). DDR mechanisms were assessed by immunostaining myocytes for an activated DNA-DSB sensor protein, phospho-H2AX (32), and a DDR effector protein, phosphorylated p53 (q-p53), which indicates p53 activation (33).…”

Section: Introductionmentioning

confidence: 99%

DNA Damage Response and DNA Repair in Skeletal Myocytes From a Mouse Model of Spinal Muscular Atrophy

Fayzullina

Martin

2016

J Neuropathol Exp Neurol

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We studied DNA damage response (DDR) and DNA repair capacities of skeletal muscle cells from a mouse model of infantile spinal muscular atrophy (SMA) caused by loss-of-function mutation of survival of motor neuron (Smn). Primary myocyte cultures derived from skeletal muscle satellite cells of neonatal control and mutant SMN mice had similar myotube length, myonuclei, satellite cell marker Pax7 and differentiated myotube marker myosin, and acetylcholine receptor clustering. DNA damage was induced in differentiated skeletal myotubes by c-irradiation, etoposide, and methyl methanesulfonate (MMS). Unexposed control and SMA myotubes had stable genome integrity. After c-irradiation and etoposide, myotubes repaired most DNA damage equally. Control and mutant myotubes exposed to MMS exhibited equivalent DNA damage without repair. Control and SMA myotube nuclei contained DDR proteins phosphop53 and phospho-H2AX foci that, with DNA damage, dispersed and then re-formed similarly after recovery. We conclude that mouse primary satellite cell-derived myotubes effectively respond to and repair DNA strand-breaks, while DNA alkylation repair is underrepresented. Morphological differentiation, genome stability, genome sensor, and DNA strand-break repair potential are preserved in mouse SMA myocytes; thus, reduced SMN does not interfere with myocyte differentiation, genome integrity, and DNA repair, and faulty DNA repair is unlikely pathogenic in SMA.

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“…1. A specific gel support is made from pipette tips from which tops are cut, yielding a small cylinder (1). These cylinders are glued side by side on the edge of a standard microscopic slide (2).…”

Section: Comet Assaymentioning

confidence: 99%

“…This size was empirically chosen so that the image easily contains one comet but is too small to contain two comets. I xy (1) where I is the intensity and x and y are positional coordinates. This projection further improves the speed of processing.…”

Section: Image Processingmentioning

confidence: 99%

Miniaturizing the comet assay with 3D vertical comets

Baert

Oostveldt

2002

Cytometry Pt A

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BackgroundThe comet or single‐cell gel electrophoresis assay is a sensitive method for the detection of DNA damage. The main drawback of comet sampling is the low cell density necessary to prevent nucleus overlap after electrophoresis, which limits large‐scale high throughput screening. Another problem may be inconsistent comet focusing. We investigated whether an approach based on three‐dimensional (3D) confocal microscopy might be beneficial for these concerns.MethodsA vertical comet assay enabling three‐dimensional confocal comet imaging of nuclei seeded at very high density was developed together with dedicated software algorithms to retrieve quantitative data at the single cell level.ResultsThree‐dimensional confocal comet imaging greatly relieved the user interactions of our nonautomated two‐dimensional comet sampling procedure. Batches of comets were blindly sampled, and confocal sectioning improved the clarity of the images and the accuracy of comet sampling. A 1‐Gy dose response was readily established. The sampling speed was competitive with that of commercial packages.ConclusionsVertical comet imaging is a new concept for fast and user‐friendly comet sampling that allows miniaturization of the assay. It may become an essential step toward high throughput screening and exploit the benefits of confocal imaging. Cytometry Part A 51A:26–34, 2003. © 2002 Wiley‐Liss, Inc.

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Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells

Cited by 1,412 publications

References 7 publications

Detection of DNA Damage in Response to Cooling Injury in Equine Spermatozoa Using Single‐Cell Gel Electrophoresis

Detection of DNA Damage in Response to Cooling Injury in Equine Spermatozoa Using Single‐Cell Gel Electrophoresis

DNA Damage Response and DNA Repair in Skeletal Myocytes From a Mouse Model of Spinal Muscular Atrophy

Miniaturizing the comet assay with 3D vertical comets

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