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 COMPUTERIZED IMAGE-ANALYSIS SYSTEM (SEESCAN)

被引：5

作者：

TAYLOR, AS

BRAUDE, PR

机构：

来源：

ZYGOTE | 1995年 / 3卷 / 01期

基金：

英国医学研究理事会;

关键词：

CELLULAR DNA CONTENT; COMPUTERIZED IMAGE ANALYSIS; DAPI; HUMAN FIBROBLASTS AND PARTHENOGENETICALLY ACTIVATED OOCYTES; MOUSE EMBRYOS;

D O I：

10.1017/S0967199400002422

中图分类号：

Q2 [细胞生物学];

学科分类号：

071009 ; 090102 ;

摘要：

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.

引用

页码：85 / 94

页数：10

共 21 条

[1]

Angell R.R., Sumner A.T., West J.D., Et al., Post-fertilisation polyploidy in human preimplantation embryos fertilised in-vitro, Hum. Reprod., 2, pp. 721-727, (1987)

[2]

Artley J., Braude P., Johnson M., Gene activity and cleavage arrest in human pre-embryos, Hum. Reprod., 7, pp. 1014-1021, (1992)

[3]

Balakier H., MacLusky N.J., Casper R.F., Characterization of the first cell cycle in human zygotes: implications for cryopreservation, Fertil. Steril., 59, pp. 359-365, (1993)

[4]

Chatelain C., Burstein S.A., Fluorescence cytophotometric analysis of megakaryocytic ploidy in culture: studies of normal and thrombocytopenic mice, Blood, 64, pp. 1193-1199, (1984)

[5]

Chisholm J.C., Johnson M.H., Warren P.D., Et al., Developmental variability within and between mouse expanding blastocysts and their ICMs, J. Embryol. Exp. Morphol., 86, pp. 311-336, (1985)

[6]

Coleman A.W., Goff L.J., Applications of fluorochromes to pollen biology, I. Mithramycin and 4',6-diamidino-2-phenylindole (DAPI) as vital stains for quantitation of nuclear DNA. Stain Technol., 60, pp. 145-154, (1985)

[7]

Coleman A.W., Maguire M.J., Coleman J.R., Mithramycin-and 4' -6' -Diamidino-2-Pheny lindole [DAPI]-DNA staining for fluoroscence microspectrophotometric measurement of DNA in nuclei, plastids, and virus particles, J. Histochem. Cytochem., 29, pp. 959-968, (1981)

[8]

Frindel E., Tubiana M., Et al., Radiobiology and the cell cycle, The Cell Cycle and Cancer, pp. 391-447, (1971)

[9]

Fulka J., Bradshaw J., Jung T., Et al., UV irradiation of metaphase II bovine oocytes (abstract). In Annual Conference of the Society for Study of Fertility, (1993)

[10]

Gentry W.L., Smith A.L., Buster J.E., Et al., Vital staining of murine blastocysts: FITC stains without decreasing viability, whereas DAPI impairs embryogenesis (abstract), (1989)

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