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PROFESSIONAL
UNIVERSITY
TERM PAPER: Fluorescence
Activated Cell Sorting (FACS)

SUBMITTED TO: Mr. Harsh


SUBMITTED BY: Charanjet yadav
SECTION: C7801
ROLL NO.: RC7801AO1
REGD NO.: 108o9907
Fluorescence Activated Cell Sorting (FACS)
Introduction:- The Fluorescence-Activated Cell Sorter (FACS) was invented in the
late 1960s by Bonner, Sweet, Hulett, Herzenberg, and others to do flow cytometry and
cell sorting of viable cells.
In multicellular organisms, all the cells are identical in their DNA but the proteins vary
tremendously. Therefore, it would be very useful if we could separate cells that are
phenotypically different from each other. In addition, it would be great to know how
many cells expressed proteins of interest, and how much of this protein they expressed.
Fluorescence Activated Cell Sorting (FACS) is a method that can accomplish all these
goals.

Principle of fluroscense activated cell sorting:-


Flow cytometry is a method used for cell-specific counting. In addition, it is
also used for
studying the expression levels of cell-surface proteins. Certain flow
cytometers also contain a fluorescence-activated cell sorter (FACS). FACS
allows for the isolation of a specific subset of cells from a mixed sample.

Procedure:- A mixed sample of cells is treated with fluorescently labeled with


specific monoclonal antibodies against a particular cell surface protein of
interest. Often this protein is a cell specific marker.

(a) If one wants to compare the expression of two different proteins, then the
same sample is treated with two different fluorescently labeled antibodies
respectively.

(b)If one wants to study the overall expression of a protein, the cells must be
treated with a mild detergent prior to antibody treatment.

The cells are suspended in a buffer such as phosphate buffer solution (PBS).
The cell suspension is forced to pass through a channel in the form of
droplets containing single cells
A laser beam is fixed down the path of the channel. As each individual cell
passes through the beam, there is a scattering of light and also an emission
from the excited fluorophores.

Both of these are detected by sensitive photomultiplier tubes.


(1) The scattering of light can be used to keep track of cell number.
(2)The fluorescence emissions indicate the expression of the targeted
protein. It indicate that how much proteins are present on cell surface.
One sorting method involves giving a charge to each droplet that passes
through the laser beam. Cells of interest are given either a positive or
negative charge, whereas cells that are not of interest are left uncharged.
Ultimately, the charged cells are passed through an electrostatic field and
directed towards tubes/plates for collection. The uncharged cells are directed
towards a waste container.
Application of fluroscent activated cell sorting:-
(a)Molecular and Cellular Analyses of Planarian Regeneration
Utilizing FACS:-
Planarians are able to regenerate their entire body structure including their brain from
any portion of their bodies utilizing pluripotent stem cells distributed throughout the
body. How can planarians maintain pluripotent stem cells through their life? How do
they regenerate complex brains from pluripotent stem cells?

To answer these questions, we utilize FACS technique. Using FACS technique we


isolate the brain neurons and stem cells for characterizing each single cell by systemic
gene expression.. Using these methods, we succeeded in isolating genes involved in
the maintenance of the stem cell state and the regulation of brain formation from
pluripotent stem cells.

(b) Use of FACS for Stem Cell Research and Regenerative Medicine
Stem cells are generally defined as clonogenic cells capable of both self-renewal and
multilineage differentiation.
The two broad types of mammalian stem cells are: embryonic stem cells that are
isolated from the inner cell mass ofblastocysts, and adult stem cells that are found in
adult tissues. In a developing embryo, stem cells can differentiate into all of the
specialized embryonic tissues. In adult organisms, stem cells and progenitor cells act as
a repair system for the body, replenishing specialized cells.
Because of these unique properties, stem cells offer the novel and exciting possibility of
organ reconstitution in place of transplanted or artificial organs in the treatment of organ
failure. therefore our aimed at prospective identification of various tissue specific stem
cells using FACS and monoclonal antibodies.
Using the FACS methodology hematopoietic stem cells of adult mouse bone marrow
cells were isolated. Use of these highly enriched HSC has enabled analysis of individual
HSC both in vivo and in vitro, contributing to better understanding of the mechanisms of
aging, self-renewal, and differentiation of HSC.
Using a similar approach, prospectively identified hepatic stem cells with multilineage
differentiation potential and self-renewing capability from fetal mouse liver.
These cells could be clonally propagated in culture for more than 6 months, where they
continuously produced hepatocytes and cholangiocytes as descendants while
maintaining a primitive stem cell subpopulation. Furthermore, upon cell transplantation,
these cells differentiated into hepato-biliary lineage cells. Manipulation of those stem
cells may provide new insights into stem cell biology as well as approaches to
regenerative medicine.
So we can say that there is a wide field of stem cells in medical
field . the stem cells of blastocyst has pluripotency nature.

This diagram show the pluripotency nature of stem cells of blastocyst.

(c) The ROCK Inhibitor Y-27632 Improves Recovery of Human


Embryonic Stem Cells after Fluorescence-Activated Cell Sorting
with Multiple Cell Surface Markers

In cell sorting of human embryonic stem cell some cell surface markers
are used like SSEA-3, TRA-1-81, and SSEA-1.
Due to the inherent sensitivity of human embryonic stem cells (hESCs) to manipulations,
the recovery and survival of hESCs after fluorescence-activated cell sorting (FACS) can be
low. The application of Y-27632 to hESCs after cell sorting improves cell recovery with no
observed effect on pluripotency, and enables the consistent recovery of hESCs by FACS
using multiple surface markers. This improved methodology for cell sorting of hESCs will aid
many applications such as removal of hESCs from secondary cell types, identification and
isolation of stem cell subpopulations, and generation of single cell clones.

(d) Antigen-specific identification and cloning of


hybridomas with a fluorescence-activated cell

Myeloma-spleen cell hybrids (hybridomas) producing antibody to mouse


immunoglobulin allotypes have been labeled with fluorescent microspheres coupled
with myeloma protein antigens. The ratio of specific to nonspecific microsphere binding
by viable hybridoma cells was about 100:1. By using a modified fluorescence-activated
cell sorter (FACS), selected hybridoma cells in a mixture have been sorted individually
into media in microculture wells, where, with thymocyte feeder cells, they developed into
clones producing a desired monoclonal antibody.