Evaluation of a simple method for measuring the cellular DNA content of mouse oocytes and embryos, human fibroblasts and parthenogenetically activated human oocytes using a computerised image analysis system (Seescan).
We report the use of a simple, reproducible, photocytometric method for measuring nuclear DNA content of DAPI-stained cells, using a computerised image analysis system: Seescan. As this technique is non-destructive and uses very short exposure to ultraviolet light, it can be used for either fixed or vital material. After correcting for any background cytoplasmic staining, the intensity of nuclear stain was measured by the Seescan and compared with that of control cells of known ploidy. Fixed material was found to stain more intensely than live material initially, but demonstrated a rapid loss of nuclear intensity over the first 90 min following removal from DAPI, after which the level plateaued. In contrast, live cells showed no change in nuclear intensity with time. The system was validated by measuring the DNA content of carefully timed mouse blastomeres, human fetal lung fibroblasts and parthenogenetically activated human oocytes. The results obtained were appropriate for the developmental stage or phenotypic appearance of each of the cell types measured.
Taylor
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AS
Assisted Conception Unit, Guy's and St Thomas' Hospital Trust, London, UK.
Braude
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PR
D000818:Animals; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis*; D004622:Embryo, Mammalian / Q000737:chemistry* / Q000166:cytology / Q000502:physiology*; D005069:Evaluation Studies as Topic; D005260:Female; D005306:Fertilization; D005333:Fetus / Q000737:chemistry; D005347:Fibroblasts / Q000737:chemistry* / Q000502:physiology*; D005456:Fluorescent Dyes; D006801:Humans; D007089:Image Enhancement; D007091:Image Processing, Computer-Assisted / Q000295:instrumentation / Q000379:methods*; D007211:Indoles / Q000737:chemistry; D008168:Lung / Q000166:cytology / Q000196:embryology; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D009865:Oocytes / Q000737:chemistry* / Q000502:physiology*; D010312:Parthenogenesis; D011247:Pregnancy; D015203:Reproducibility of Results; D015842:Serine Proteinase Inhibitors; D013997:Time Factors; D014466:Ultraviolet Rays
D005456:Fluorescent Dyes; D007211:Indoles; D015842:Serine Proteinase Inhibitors; C007293:DAPI; D004247:DNA