This document provides information about two molecular biology techniques: fluorescence-activated cell sorting (FACS) and fluorescence in situ hybridization (FISH). FACS uses fluorescent markers and antibodies to isolate specific cell populations based on cell surface markers. FISH detects and locates DNA sequences on chromosomes using fluorescent probes. It attaches fluorescent dyes to DNA probes that bind to matching sequences on chromosomes, allowing their visualization. Both techniques are important tools in research areas like cancer research, stem cell biology, and hematology.

1. FACS (FLUORESCENCE-ACTIVATED CELL
SORTING) & FISH (FLUORESCENCE IN
SITU HYBRIDIZATION)
PRESENTED BY
S. MOHANA
II PG BIOTECHNOLOGY

2. FLUORESCNECE ACTIVATED CELL SORTING (FACS)
INTRODUCTION
 Fluorescence-activated cell sorting (FACS), sometimes called
fluorescence-assisted cell sorting, is a specialized type of flow
cytometry that uses fluorescent markers to target and isolate cell
groups. This cell sorting technique is commonly used in
hematopoiesis, oncology, and stem cell biology research.
 FACS technology separates cells based on cell surface markers.
Antigenic ligands, such as proteins and carbohydrates, give each cell a
unique surface phenotype, and specific antibodies associated with the
cell surface antigens are then used to target cells with those antigens.

3. PRINCIPLE
 The basic principle of flow cytometry is the
passage of cells in single file in front of a laser
so they can be detected, counted and sorted.
Cell components are fluorescently labelled and
then excited by the laser to emit light at varying
wavelengths. detected by the detectors.

4. WORK FLOW OF FACS

5. APPLICATIONS OF FACS
 Immuno phenotyping
 Cell Sorting
 Cell Cycle Analysis
 Apoptosis
 Cell Proliferation Assays
 Intracellular Calcium Flux

6. FLUORESCENCE IN SITU HYBRIDIZATION- FISH
INTRODUCTION
 Fluorescence in situ hybridization (abbreviated FISH) is a laboratory technique
used to detect and locate a specific DNA sequence on a chromosome.
 In this technique, the full set of chromosomes from an individual is affixed to a
glass slide and then exposed to a “probe”—a small piece of purified DNA tagged
with a fluorescent dye. The fluorescently labeled probe finds and then binds to its
matching sequence within the set of chromosomes. With the use of a special
microscope, the chromosome and sub-chromosomal location where the fluorescent
probe bound can be seen.

In this approach, a fluorescent dye is attached to a purified piece of DNA, and
then that DNA is incubated with the full set of chromosomes from the originating
genome, which have been attached to a glass microscope slide. The fluorescently
labeled DNA finds its matching segment on one of the chromosomes, where it
sticks

7. PRINCIPLE
 The basic principle involved is hybridization of nuclear DNA of either
interphase cells or of metaphase chromosomes affixed to a
microscopic slide, with a nucleic acid probe. The probes are either
labeled indirectly with a hapten or directly through incorporation of a
fluorophore.

9. APPLICATIONS
 Histiocytoid Sweet Syndrome -Here FISH was performed to determine the
presence of BCR/ABL gene fusion
 Differential Diagnosis of Pseudomosaicism from True Mosaicism
 Autologous Fat Grafting - FISH can be used to detect specific DNA or
ribonucleic acid (RNA) sequences within the context of the cell
 Dedifferentiated Liposarcoma (DDLPS)
 Streptococcus Pneumonia - can be detected through FISH from blood culture
samples
 Aneuploidies - With the help of FISH, the detection of chromosome signals in
interphase nuclei is possible

10. Application of FISH in oncology
 Chronic Myeloid Leukemia (CML) - The BCR/ABL1 translocation often occurs in
chronic myeloid leukemia (CML). This gene was detected by FISH.
 Multiple Myelomas (MM)
 Pulmonary Adenocarcinomas - The BCR/ABL1 translocation often occurs in
chronic myeloid leukemia (CML)
 Prostate Cancer - FISH assay was used for the detection of either TMPRSS2 or
ERG rearrangements.
 Breast Carcinomas - The FISH assay is regarded as a vital tool for clinical
evaluation of the HER2 status.

11.  Cholangiocarcinoma (CC)
 Melanoma

12. THANK YOU