پديد آورندگان :
Mortaza Hosseini Sayyed Mortaza نويسنده , Hajian Mehdi نويسنده , Asgari Vajihe نويسنده , Forozanfar Mohsen نويسنده , Abedi Parvaneh نويسنده , NASR ESFAHANI MOHAMMAD HOSSEIN نويسنده
چكيده لاتين :
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; 300llg/ml)
and bisbenzimide (Hoechst: H33342; Sug/ml) fluorescent dyes. Embryos were then freed
from residual dyes by thoroughly washing in warm phosphate buffer saline free ofcalcium 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.
