- Cell synchronization
Cell Synchronization. Synchronization literally means to make two or more things happen exactly simultaneously. For instance, two or more watches can be synchronized to show exactly the same time.The cells at different stages of the cell cycle in a culture can be synchronized so that the cells will be at the same phase. Cell synchrony is required to study the progression of cells through cell cycle. They are broadly categorized into two groups - Physical Fractionation for Cell Separation and Chemical Blockade for Cell Separation.
Cell separation by physical means
Physical fractionation or cell separation techniques, based on the following characteristics are in use.
*Affinity of antibodies on cell surface
*Light scatter or
fluorescentemission by labeled cells.The two commonly used techniques are:
The physical characteristics- cell size and sedimentation velocity are operative in the technique of centrifugal
elutriation. Centrifugal elutriator (from Beckman) is an advanced device for increasing the sedimentation rate so that the yield and resolution of cells is better. The cell separation is carried out in a specially designed centrifuge and rotor.
Fluorescence-activated cell sorting
Fluorescence-activated cell sorting is a technique for sorting out the cells based on the differences that can be detected by light scatter (eg. cell size) or fluorescence emission (by penetrated DNA, RNA, proteins, antigens).The procedure involves passing of a single stream of cells through a
laserbeam so that the scattered light from the cells can be detected and recorded. There are two instruments in use based on its principle:
* b) Fluorescent-Activated Cell Sorter (FACS)
Cell separation by chemical blockade
The cells can be separated by blocking metabolic reactions. Two types of metabolic blockades are in use:
Inhibition of DNA synthesis
During the S Phase of cell cycle, DNA synthesis can be inhibited by using inhibitors such as thymidine, aminopterin, hydroxyurea and cytosine arabinoside. The effects of these inhibitors are variable. The cell cycle is predominantly blocked in S phase that results in Viable Cells.
Elimination of serum from the culture medium for about 24 hours results in the accumulation of cells at G1 phase. This effect of nutritional deprivation can be restored by their addition by which time the cell synchrony occurs.
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