FLUROSCENT ACTIVATED CELL SORTER

FLUROSCENT ACTIVATED CELL SORTER

10.      FLUROSCENT ACTIVATED CELL SORTER

Fluroscent Activated Cell Sorter enable a mixture of different cells, to be shorter into different containers.
Cells are sorted according to their specific light scattering and Furious and characteristic.
Principle:-
An antibody specific for a particular cell surface protein is associated with a fluorescent molecule and then add to a mixture of cells.
Fluorescent monitored when the specific cells pass through the laser beam. Droplets containing single cells are giving positive or negative charge based on the number of fluorescent antibodies. 
Droplets are then detected by the electric field into collection tube according to their charge.

Applications- is in the number of fields - molecular Biology, immunology, pathology, virology, plant and Marine biology.
-    In medicine \rightarrow hematology, tumour immunology, transplantation, parental diagnosis, sperm abnormality.
-    Apoptosis and DNA damage. 
-    In microbiology, it can be used to and sort mutant libraries.
Forward scatter and side 2D scatter - 
Scattered light is measured by two  optical detector
1.     One detects major scatter light along the path of the laser- this is referred to as forward scatter (FSC).
2.    The other detector measures scatter light at an angle 90 degree relative to the laser- this is referred to as side scatter (SSC). 
Forward Scattering- Forward Scattering intensity is propositional to the detector of the cell and is primarily due to light diffraction around the cell.
*     Measurement of forwarding scattering allows for the discrimination of cells by size.
*     Forward scatter \rightarrow detected by the photodiode, weed control the light into electrical signal.
*     The voltage hence produced is proportional to the diameter of the cell.
*     Forward scatter is very helpful for differentiating cell of the immune system.
Side 2-D scattering \rightarrow This is for information about the internal complexity of the cell. The interface between the laser and internal structure cause the light to reflect on refract.
-    Cellular components that are Scatter include granules and the nucleus.
-     Relative forward scatter, light signals from side scatter are weak.
*     A photomultiplier tube (PMT) is used to measure side scatter become it is a more sensitive optical detector.
*     Side scatter is helpful for the identification of a cell with varying complexity. 
*     Each signal with passes into a detector (PMT or photodiode) \rightarrow record characters.
-    Within scatter parameters, pulse height v/s pulse width plate are used to isolate single cells passing through the cytometer.


Application of FACS:-
1)     Cell sorting on the Basis of the presence of Receptor:-
Cells can be sort on the basis of receptor present on it. For example, we took a mixture of the cells containing 4 types of T cell (A, B, C, D).
Cell A contains no receptor.
Cell B contain receptor of CD4.
Cell C contain receptor of CD8.
Cell D contain receptor of both CD4, CD8.
The fluorescent label Ab against CD4 (FITC) & CD8 ( Rodhamine) receptor were added in the mixture of cells to allow the binding of Ab with cell receptor then the sample was sorted in flow cytometry. A quartet graph was plotted and analysed.


Quartet (Q1, Q2, Q3, Q4)
Q1 show low signal of FITC (Ab- CD 4) and high signal for Rodhamine. Q2 shows the high signal of FITC & Rodhamine, Q3 Show low signal for FITC & Rodhamine. Q4 show high signal for FITC & low signal for Rodhamine.


1)     In quartet Q1 cells have High signals for Rodhamine, these are cell C containing receptor of CD8.
2)     In quartet Q2 cells have High signal for both FITC & Rodhamine. These are cell D with the receptor of both CD4     & CD8.
3)     In quartet Q3 cells have the low signal for both Rodhamine & FITC. These are cell A with no receptor.
4)     In quartet Q4 cells have High signal for FITC. These are cell B with the receptor of CD4.
Sorting of Blood cells (Granulocyte & Agranulocyte) :
With the help of FACS, we can sort blood cells on the basis of scattering of the laser, Forward scattering and side scattering. 
There are two types of white blood cells  Granulocyte & Agranulocyte.
A Granulocyte show forward scattering of laser and less side scattering or no side scattering of the laser. Granulocyte shows a more side scattering of laser and less forward scattering.
Small size cell show less forward scattering of laser or large size cell show more forward scattering of the laser.
Cell sample loaded in flow cytometry for analysis and sorting on the basis of forwarding scattering and side scattering of the laser, and analysis it by the quartet graph.


Analysis:-
In the quartet, Q1 Forward scattering is less which indicate small size of the cell and more side scattering indicates granulocyte. Small size granulocyte is acidophilus.
In the quartet, Q2 Forward scattering is more which indicate the large size of the cell and more side scattering it indicates granulocyte. Large size granulocyte is neutrophils.
In quartet Q3  forward scattering is less which indicate the small size and less side scattering which show cell is agranulocyte. Small size agranulocyte is lymphocytes.
In quartet Q4 forward scattering is more which indicates the large size of the cell and less side scattering which show cell is agranulocyte. Large size agranulocyte is monocytes.
On the basis of the phase of cells:-
Calls can be sort on the basis of the phase of the cell cycle at which cell is present.
For sorting of different phase cells, we use the Hoechst stain. Hoechst stain binds wit Double-stranded DNA it a fluorescent dye.

The cell in G1 phase have less amount of DNA, in S phase cell have more amount of double-strand DNA but less than G2 phase. G2 phase cell has more amount of double strand DNA. More amount of double-strand DNA show more fluorescence. Hoechst stain mixed with the mixture of cells to allow stain bind with double strand DNA. Cell mixture analysed by flow cytometry. A line diagram graph shows the amount of cell in different stages by fluorescence.


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