A brief outline of the procedural cytogenetic steps
ساعت ۱:۱۸ ‎ب.ظ روز ۱۳۸٩/٤/٩  کلمات کلیدی:

 

Cell culture:

Although cell culture methods vary significantly with the tissue of origin (amniotic fluid, chorionic villi, fetal tissues, peripheral blood, bone marrow, solid tumors, cell lines of various origins), the final goal is to achieve cell growth and division, ultimately leading to a good mitotic index.


Harvesting:

1.     Mitotic spindle formation is blocked, usually by adding colcemide to the culture, and the cell division is stopped at the metaphase level.

2.     Cells are subjected to hypotonic treatment, which increases their volume, and disrupts the cell membrane of the red blood cells allowing their removal.

3.     A fixative solution is added to the cell suspension to preserve the cells in their "swollen" state and to remove the water, thus "hardening" the biologic material.

4.     The common fixative (3:1 methanol:acetic acid) removes lipids and alters/denatures proteins thus making the cell membrane remainder very fragile, which is important for subsequent chromosome spreading.

 

Slide preparation:

Drops of cell suspension are placed on a slide, and allowed to dry in a controlled fashion, leading to chromosome spreading.


Aging:

Slides are subjected to dry heat and/or ethanol, in order to:

1.     denature the proteins,

2.     remove water and fixative from the preparations, and

3.     enhance the adherence of the material to the glass.

 

Banding:  

Spreaded chromosomes are stained/banded, which allows their visualization and identification at the microscope[1]



[1] http://info.med.yale.edu/genetics/ward/tavi/