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International Journal of Fertility and Sterility، جلد ۱، شماره ۳، صفحات ۱۰۳-۱۰۶
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عنوان انگلیسی Novel Approach of Differential Staining to Detect Necrotic Cells in Preimplantation Embryos
چکیده انگلیسی مقاله Background: This novel approach describes a rapid and simple method for identification of necrotic vs. viable cells within a mammalian blastocyst.
Materials and Methods: Hatched bovine blastocysts produced in vitro were first incubated for 30 min in pre-equilibrated culture medium containing propidium iodide (PI; 300μg/ml) and bisbenzimide (Hoechst: H33342; 5μg/ml) fluorescent dyes. Embryos were then freed from residual dyes by thoroughly washing in warm phosphate buffer saline free of calcium and magnesium (PBS-), fixed in 2.5% glutharaldehyde and washed again in PBS- . Stained embryos afterwards were mounted in a drop of glycerol over a microscopic slide. Prepared samples were examined under an epifluorescent microscope using the same excitation wavelength (330-385nm) and barrier filter (400nm) to distinguish necrosed vs. viable blastomers as being appeared in red and blue, respectively.
Results: Obtained results showed that in cells with altered cell membrane such as late apoptotic or necrotic cells, PI and H33342 readily enter through the cytoplasmic barriers and so the chromatin materials are stained by both, but since PI quenches bisbenzimide fluorescence, necrotic blastomeres are seen in red to pinky red, while live cells are seen just as blue.
Conclusion: Obtained results clearly indicated that this novel approach can be used as a simple, feasible and precise method for every embryology lab and with all the mammalian blastocysts produced either in vitro or in vivo. The basic assay can be completed in 60 min, and valuable and reliable information can be obtained about the quality of the embryos.
کلیدواژه‌های انگلیسی مقاله Mammalian Blastocyst, Viability, Hoechst 33342, Propidium Iodide
نویسندگان مقاله Seyyed Morteza Hosseini |
Clinical and Experimental Embryology Department, Royan Institute

Mehdi Hajian |
Clinical and Experimental Embryology Department, Royan Institute

Vajihe Asgari |
Anatomy Department, Isfahan University of Medical Sciences

Mohsen Forozanfar |
Faculty of Basic Science, Islamic Azad University, Marvdasht Branch

Parvaneh Abedi |
Clinical and Experimental Embryology Department, Royan Institute

Mohammad Hossein Nasr-Esfahani |
Clinical and Experimental Embryology Department, Royan Institute

نشانی اینترنتی https://www.ijfs.ir/article_45652_f551dc5119a235e9ff2651ef3179bb08.pdf
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