Separating the cell of one type from the pool of heterogeneous cell population so that we can use them to develop culture of cells to know much more information about that kind of cell is called cell sorting.
Methods of Cell Sorting:
1) Surface Adhesion: Different types of cells have different affinities towards a solid surface in presence of varying quantities of enzymes. This property can be exploited to separate cells.
2) Centrifugation: In a centrifuge tube, larger cells settle at the bottom and smaller cells remain suspended, thus separating them.
3) Fluorescent Activated Cell Sorting: It is the most sophisticated cell-separation method or technique uses an antibody coupled to a fluorescent dye to label
specific cells. The labeled cells can then be separated from the unlabelled ones in an electronic fluorescence-activated cell sorter.
Some facts about FACS:
a) First sorter was invented in 1965 by Mack Fowler
b) It provides the method of sorting a heterogeneous mixture of cells into two or more containers based on fluorescent character of each cell
c) Uses antibody coupled to a fluorescent dye to label specific cells
d) It can separate 1 fluorescent cell from a poll of 1000 unlabeled cells
e) It can separate 300000 cells per minute
Fluorescence-activated cell sorting is a specialized type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. It is a useful scientific instrument as it provides fast, objective and quantitative recording of fluorescent signals from individual cells as well as physical separation of cells of particular interest. The acronym FACS is trademarked and owned by Becton Dickinson.
Major components of FACS are :
i) Sheath fluid: The liquid stream (sheath fluid) carries and aligns the cells so that they pass single file through the light beam for sensing. Generally PBS or Tris-Cl are used for this purpose as the are isotonic with the cells.
ii) Vibrating nozzle: It breaks the cell suspension into fine droplets
iii) Laser: To illuminate the droplets
iv) Detector: To detect the resulting fluorescence .It can be either a CCD or PMT.
v) Analyzer: It measures the fluorescence and decides what charge is to be given.
vi) Charger: Gives a specific electric charge to the cells passing through.
Process of Operation:
* The cell suspension containing the cells labeled with fluorescent dye is directed into a thin stream so that all cells pass in a single file
* The stream emerges from nozzle vibrating at a some 40000cps
* It breaks the stream into 40000 droplets per sec
* Some of these droplet may containing cell
* Laser beam is directed at the stream just before it breaks up into droplets
* As each labeled cell passes through the beam, its resulting fluorescence is detected by a photocell
* If the cell is fluorescent then it is given a charge +ve or -ve
* The droplets retain this charge as they pass between a pair of charged metal plates
* Positively charged cells are attracted by negatively charged plate and vice versa
* Uncharged droplet doesn't deviate and it pass straight into the third container and discarded later
Data Representation by Computers:
1) The intensity of the green or red fluorescence is plotted on the X-axis and the number of cells with each level of fluorescence is plotted on the Y-axis
2) This method is best if all cells are either green, red or unlabeled and no cells are labeled both colors
The X-axis plots the intensity of green fluorescence while the Y-axis plots the intensity of red fluorescence. The individual black dots represent individual cells and
we are not supposed to count the dots but just look at the relative density of dots in each quadrant.
Different types of Lasers used in FACS:
• Blue argon laser(488nm)
• UV He-Cd laser(325nm)
• UV lamp(366nm)-used for DNA work
• Violet diode laser(405nm)
• Green He-Ne laser(543nm)
Commonly Used Fluorochromes:
• Lucifer yellow
• Texas Red
• Cascade Blue
• Alexa Flour (20 colors)
• Ethidium Bromide
• Chromomycin A3
Uses of FACS:
a) Counting T and B cells
b) Measuring Cell Permeability
c) Immunophenotyping (cell surface antigens, intracellular antigens, etc.)
d) Detecting transgenic products like GFP in cell
e) Chromosome analysis and sorting
f) Differentiating cancerous cells
g) Analysis of intracellular ion concentration
h) Cell cycle analysis (by using DNA-binding dye)
i) Multiplex proteins assay (cytometric bead array)
j) Characterization of multi-drug resistant (MDR) cells
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