This document provides an overview of flow cytometry. It begins by defining flow cytometry as a technique for quantitative single cell analysis that counts, examines, and sorts cells based on optical properties like light scattering and fluorescence. It then describes the basic principles, components, and working of a flow cytometer. Key components include lasers, optical filters, and detectors. Cells in suspension pass through the laser one by one, with signals detected by photodiodes and photomultiplier tubes. Applications discussed include cell sorting, apoptosis analysis using markers like Annexin V and PI, and cell cycle analysis using DNA binding dyes or BrdU incorporation. Clinical uses involve hematologic malignancy diagnosis, residual disease detection, and monitoring treatments.
Fluorescence- Activated Cell Sorter is a powerful technique used in cell sorting, cell-cycle analysis etc.
The presentation gives a basic understanding of the principle of FACS, instrumentation, interpretation of results, applications, how to do cell-cycle analysis using FACS and various troubleshooting tips.
FLOW CYTOMETRY, PRINCIPLE, APPLICATION, USE IN HAEMATOLOGY, COMPONENT OF FLOW CYTOMETRY, DATA INTERPRETATION, DATA ANALYSIS, CELL SHORTING ADVANTAGES AND DISADVANTAGES, IMMUNOLOGICAL CLASSIFICATION OF ACUTE
LEUKEMIA
Fluorescent in situ hybridization (FISH) is a cytogenetic technique that can be used to detect and localize the presence or absence of specific DNA sequences on chromosomes.
It is a laser based technology that measures and analyses different physical and chemical properties of the cells/particles flowing in a stream of fluid through a beam of light
A fluorescence microscope is an optical microscope that uses fluorescence and phosphorescence instead of, or in addition to, reflection and absorption to study properties of organic or inorganic substances.
Fluorescence- Activated Cell Sorter is a powerful technique used in cell sorting, cell-cycle analysis etc.
The presentation gives a basic understanding of the principle of FACS, instrumentation, interpretation of results, applications, how to do cell-cycle analysis using FACS and various troubleshooting tips.
FLOW CYTOMETRY, PRINCIPLE, APPLICATION, USE IN HAEMATOLOGY, COMPONENT OF FLOW CYTOMETRY, DATA INTERPRETATION, DATA ANALYSIS, CELL SHORTING ADVANTAGES AND DISADVANTAGES, IMMUNOLOGICAL CLASSIFICATION OF ACUTE
LEUKEMIA
Fluorescent in situ hybridization (FISH) is a cytogenetic technique that can be used to detect and localize the presence or absence of specific DNA sequences on chromosomes.
It is a laser based technology that measures and analyses different physical and chemical properties of the cells/particles flowing in a stream of fluid through a beam of light
A fluorescence microscope is an optical microscope that uses fluorescence and phosphorescence instead of, or in addition to, reflection and absorption to study properties of organic or inorganic substances.
Flow cytometry is a technique used in Cell Biology to analyze and measure the volume of cells suspended in a liquid with streamline flow, exposed to a laser beam.
Flow cytometry (FCM) is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles. In this process, a sample containing cells or particles is suspended in a fluid and injected into the flow cytometer instrument.
The technique of flow cytometry is used to evaluate cells for a number of functions, such as cell counting, phenotyping, cell cycle analysis, and viability.
Gene Therapy, Somatic cell gene therapy, germ line gene therapy, classical gene therapy, non-classical gene therapy, targets of gene therapy, barriers of gene therapy, ex vivo gene therapy, in vivo gene therapy, vectors for gene delivery, antisense therapy
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Ve...kevinkariuki227
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Verified Chapters 1 - 19, Complete Newest Version.pdf
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Verified Chapters 1 - 19, Complete Newest Version.pdf
MANAGEMENT OF ATRIOVENTRICULAR CONDUCTION BLOCK.pdfJim Jacob Roy
Cardiac conduction defects can occur due to various causes.
Atrioventricular conduction blocks ( AV blocks ) are classified into 3 types.
This document describes the acute management of AV block.
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Tom Selleck Health: A Comprehensive Look at the Iconic Actor’s Wellness Journeygreendigital
Tom Selleck, an enduring figure in Hollywood. has captivated audiences for decades with his rugged charm, iconic moustache. and memorable roles in television and film. From his breakout role as Thomas Magnum in Magnum P.I. to his current portrayal of Frank Reagan in Blue Bloods. Selleck's career has spanned over 50 years. But beyond his professional achievements. fans have often been curious about Tom Selleck Health. especially as he has aged in the public eye.
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Introduction
Many have been interested in Tom Selleck health. not only because of his enduring presence on screen but also because of the challenges. and lifestyle choices he has faced and made over the years. This article delves into the various aspects of Tom Selleck health. exploring his fitness regimen, diet, mental health. and the challenges he has encountered as he ages. We'll look at how he maintains his well-being. the health issues he has faced, and his approach to ageing .
Early Life and Career
Childhood and Athletic Beginnings
Tom Selleck was born on January 29, 1945, in Detroit, Michigan, and grew up in Sherman Oaks, California. From an early age, he was involved in sports, particularly basketball. which played a significant role in his physical development. His athletic pursuits continued into college. where he attended the University of Southern California (USC) on a basketball scholarship. This early involvement in sports laid a strong foundation for his physical health and disciplined lifestyle.
Transition to Acting
Selleck's transition from an athlete to an actor came with its physical demands. His first significant role in "Magnum P.I." required him to perform various stunts and maintain a fit appearance. This role, which he played from 1980 to 1988. necessitated a rigorous fitness routine to meet the show's demands. setting the stage for his long-term commitment to health and wellness.
Fitness Regimen
Workout Routine
Tom Selleck health and fitness regimen has evolved. adapting to his changing roles and age. During his "Magnum, P.I." days. Selleck's workouts were intense and focused on building and maintaining muscle mass. His routine included weightlifting, cardiovascular exercises. and specific training for the stunts he performed on the show.
Selleck adjusted his fitness routine as he aged to suit his body's needs. Today, his workouts focus on maintaining flexibility, strength, and cardiovascular health. He incorporates low-impact exercises such as swimming, walking, and light weightlifting. This balanced approach helps him stay fit without putting undue strain on his joints and muscles.
Importance of Flexibility and Mobility
In recent years, Selleck has emphasized the importance of flexibility and mobility in his fitness regimen. Understanding the natural decline in muscle mass and joint flexibility with age. he includes stretching and yoga in his routine. These practices help prevent injuries, improve posture, and maintain mobilit
NVBDCP.pptx Nation vector borne disease control programSapna Thakur
NVBDCP was launched in 2003-2004 . Vector-Borne Disease: Disease that results from an infection transmitted to humans and other animals by blood-feeding arthropods, such as mosquitoes, ticks, and fleas. Examples of vector-borne diseases include Dengue fever, West Nile Virus, Lyme disease, and malaria.
Title: Sense of Smell
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the primary categories of smells and the concept of odor blindness.
Explain the structure and location of the olfactory membrane and mucosa, including the types and roles of cells involved in olfaction.
Describe the pathway and mechanisms of olfactory signal transmission from the olfactory receptors to the brain.
Illustrate the biochemical cascade triggered by odorant binding to olfactory receptors, including the role of G-proteins and second messengers in generating an action potential.
Identify different types of olfactory disorders such as anosmia, hyposmia, hyperosmia, and dysosmia, including their potential causes.
Key Topics:
Olfactory Genes:
3% of the human genome accounts for olfactory genes.
400 genes for odorant receptors.
Olfactory Membrane:
Located in the superior part of the nasal cavity.
Medially: Folds downward along the superior septum.
Laterally: Folds over the superior turbinate and upper surface of the middle turbinate.
Total surface area: 5-10 square centimeters.
Olfactory Mucosa:
Olfactory Cells: Bipolar nerve cells derived from the CNS (100 million), with 4-25 olfactory cilia per cell.
Sustentacular Cells: Produce mucus and maintain ionic and molecular environment.
Basal Cells: Replace worn-out olfactory cells with an average lifespan of 1-2 months.
Bowman’s Gland: Secretes mucus.
Stimulation of Olfactory Cells:
Odorant dissolves in mucus and attaches to receptors on olfactory cilia.
Involves a cascade effect through G-proteins and second messengers, leading to depolarization and action potential generation in the olfactory nerve.
Quality of a Good Odorant:
Small (3-20 Carbon atoms), volatile, water-soluble, and lipid-soluble.
Facilitated by odorant-binding proteins in mucus.
Membrane Potential and Action Potential:
Resting membrane potential: -55mV.
Action potential frequency in the olfactory nerve increases with odorant strength.
Adaptation Towards the Sense of Smell:
Rapid adaptation within the first second, with further slow adaptation.
Psychological adaptation greater than receptor adaptation, involving feedback inhibition from the central nervous system.
Primary Sensations of Smell:
Camphoraceous, Musky, Floral, Pepperminty, Ethereal, Pungent, Putrid.
Odor Detection Threshold:
Examples: Hydrogen sulfide (0.0005 ppm), Methyl-mercaptan (0.002 ppm).
Some toxic substances are odorless at lethal concentrations.
Characteristics of Smell:
Odor blindness for single substances due to lack of appropriate receptor protein.
Behavioral and emotional influences of smell.
Transmission of Olfactory Signals:
From olfactory cells to glomeruli in the olfactory bulb, involving lateral inhibition.
Primitive, less old, and new olfactory systems with different path
Flu Vaccine Alert in Bangalore Karnatakaaddon Scans
As flu season approaches, health officials in Bangalore, Karnataka, are urging residents to get their flu vaccinations. The seasonal flu, while common, can lead to severe health complications, particularly for vulnerable populations such as young children, the elderly, and those with underlying health conditions.
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Protect yourself and your loved ones by getting vaccinated. Together, we can help keep Bangalore healthy and safe this flu season. For more information on vaccination centers and schedules, residents can visit the Karnataka Health Department’s official website or follow their social media pages.
Stay informed, stay safe, and get your flu shot today!
New Directions in Targeted Therapeutic Approaches for Older Adults With Mantl...i3 Health
i3 Health is pleased to make the speaker slides from this activity available for use as a non-accredited self-study or teaching resource.
This slide deck presented by Dr. Kami Maddocks, Professor-Clinical in the Division of Hematology and
Associate Division Director for Ambulatory Operations
The Ohio State University Comprehensive Cancer Center, will provide insight into new directions in targeted therapeutic approaches for older adults with mantle cell lymphoma.
STATEMENT OF NEED
Mantle cell lymphoma (MCL) is a rare, aggressive B-cell non-Hodgkin lymphoma (NHL) accounting for 5% to 7% of all lymphomas. Its prognosis ranges from indolent disease that does not require treatment for years to very aggressive disease, which is associated with poor survival (Silkenstedt et al, 2021). Typically, MCL is diagnosed at advanced stage and in older patients who cannot tolerate intensive therapy (NCCN, 2022). Although recent advances have slightly increased remission rates, recurrence and relapse remain very common, leading to a median overall survival between 3 and 6 years (LLS, 2021). Though there are several effective options, progress is still needed towards establishing an accepted frontline approach for MCL (Castellino et al, 2022). Treatment selection and management of MCL are complicated by the heterogeneity of prognosis, advanced age and comorbidities of patients, and lack of an established standard approach for treatment, making it vital that clinicians be familiar with the latest research and advances in this area. In this activity chaired by Michael Wang, MD, Professor in the Department of Lymphoma & Myeloma at MD Anderson Cancer Center, expert faculty will discuss prognostic factors informing treatment, the promising results of recent trials in new therapeutic approaches, and the implications of treatment resistance in therapeutic selection for MCL.
Target Audience
Hematology/oncology fellows, attending faculty, and other health care professionals involved in the treatment of patients with mantle cell lymphoma (MCL).
Learning Objectives
1.) Identify clinical and biological prognostic factors that can guide treatment decision making for older adults with MCL
2.) Evaluate emerging data on targeted therapeutic approaches for treatment-naive and relapsed/refractory MCL and their applicability to older adults
3.) Assess mechanisms of resistance to targeted therapies for MCL and their implications for treatment selection
ARTIFICIAL INTELLIGENCE IN HEALTHCARE.pdfAnujkumaranit
Artificial intelligence (AI) refers to the simulation of human intelligence processes by machines, especially computer systems. It encompasses tasks such as learning, reasoning, problem-solving, perception, and language understanding. AI technologies are revolutionizing various fields, from healthcare to finance, by enabling machines to perform tasks that typically require human intelligence.
Title: Sense of Taste
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the structure and function of taste buds.
Describe the relationship between the taste threshold and taste index of common substances.
Explain the chemical basis and signal transduction of taste perception for each type of primary taste sensation.
Recognize different abnormalities of taste perception and their causes.
Key Topics:
Significance of Taste Sensation:
Differentiation between pleasant and harmful food
Influence on behavior
Selection of food based on metabolic needs
Receptors of Taste:
Taste buds on the tongue
Influence of sense of smell, texture of food, and pain stimulation (e.g., by pepper)
Primary and Secondary Taste Sensations:
Primary taste sensations: Sweet, Sour, Salty, Bitter, Umami
Chemical basis and signal transduction mechanisms for each taste
Taste Threshold and Index:
Taste threshold values for Sweet (sucrose), Salty (NaCl), Sour (HCl), and Bitter (Quinine)
Taste index relationship: Inversely proportional to taste threshold
Taste Blindness:
Inability to taste certain substances, particularly thiourea compounds
Example: Phenylthiocarbamide
Structure and Function of Taste Buds:
Composition: Epithelial cells, Sustentacular/Supporting cells, Taste cells, Basal cells
Features: Taste pores, Taste hairs/microvilli, and Taste nerve fibers
Location of Taste Buds:
Found in papillae of the tongue (Fungiform, Circumvallate, Foliate)
Also present on the palate, tonsillar pillars, epiglottis, and proximal esophagus
Mechanism of Taste Stimulation:
Interaction of taste substances with receptors on microvilli
Signal transduction pathways for Umami, Sweet, Bitter, Sour, and Salty tastes
Taste Sensitivity and Adaptation:
Decrease in sensitivity with age
Rapid adaptation of taste sensation
Role of Saliva in Taste:
Dissolution of tastants to reach receptors
Washing away the stimulus
Taste Preferences and Aversions:
Mechanisms behind taste preference and aversion
Influence of receptors and neural pathways
Impact of Sensory Nerve Damage:
Degeneration of taste buds if the sensory nerve fiber is cut
Abnormalities of Taste Detection:
Conditions: Ageusia, Hypogeusia, Dysgeusia (parageusia)
Causes: Nerve damage, neurological disorders, infections, poor oral hygiene, adverse drug effects, deficiencies, aging, tobacco use, altered neurotransmitter levels
Neurotransmitters and Taste Threshold:
Effects of serotonin (5-HT) and norepinephrine (NE) on taste sensitivity
Supertasters:
25% of the population with heightened sensitivity to taste, especially bitterness
Increased number of fungiform papillae
Knee anatomy and clinical tests 2024.pdfvimalpl1234
This includes all relevant anatomy and clinical tests compiled from standard textbooks, Campbell,netter etc..It is comprehensive and best suited for orthopaedicians and orthopaedic residents.
3. 1. Introduction
• Flow cytometry is a technique of quantitative single cell analysis.
• This technique was first described by Wallace Coulter in the 1950s.
• The flow cytometer was developed in the 1970’s and applied to
automated cell counting.
• Flow cytometer count, examine and sort cells based on their optical
properties (Scattering and fluorescence).
• The present “state-of-the-art” flow cytometers are capable of
analyzing upto 13 parameters (forward scatter, side scatter, 11 colors
of immunofluorescence).
5. 2. Basic Principle of Flow Cytometry
• Prepared single cell or particle
suspension is necessary for flow
cytometric analysis.
• The suspension of cells or particles
is aspirated into a channel
surrounded by a narrow fluid
system.
• They pass one at a time through a
focused laser beam.
• The light is either scattered or
absorbed when it strikes a cell.
6. • Light scattering is dependent on
the internal structure of the cell
and its size and shape.
• Absorbed light of the appropriate
wavelength may be re-emitted as
fluorescence. (The cell may have a
naturally fluorescent substance or
one or more fluorochrome-labelled
antibodies are attached to surface
or internal cell structures).
7. • Light and/or fluorescence scatter signals are detected by a series of
photodiodes and amplified.
• Optical filters are essential to block unwanted light and permit light of
the desired wavelength to reach the photodetector.
• Fluorescein isothiocynate (FITS), Texas red and phycoerythrin (PE) are
the most common fluorescent dyes used in the biomedical sciences.
• Large number of cells are analysed in a short period of time
(>1,000/sec).
9. 3. Working of Flow Cytometer
• A flow cytometer is composed of three main systems:
1. Fluidics – Transport cells in a stream to the laser beam for interrogation.
2. Optics – Consist of lasers to illuminate the cells in the sample stream and
optical filters to direct the resulting light signals to the appropriate
detectors.
3. Electronics – Converts the detected light signals into electrical signals that
can be processed by computer.
10. 3.1 Fluidics System
• Flow cytometers use the
principle of hydrodynamic
focusing for presenting cells one
at a time to a light source.
• The fluidics system consist of a
central channel through which
the sample is injected, enclosed
by an outer sheath that contains
faster flowing fluid.
11. • As the sheath fluid moves, it creates a massive drag effect on the
narrowing central chamber. This alters the velocity of the central fluid.
• The velocity of the central fluid becomes parabolic i.e., greatest
velocity at the center and zero velocity at the wall..
• This effect creates a single stream of particles or cells and is called
hydrodynamic focusing.
• Under laminar flow conditions, the fluid in the central chamber will
not mix with sheath fluid.
12. 3.2 Optics
• Light scattering or fluorescence
emission provides information
about the cell’s properties.
• Light that is scattered by an object
is detected by different detectors.
• One detector is placed in line with
the beam to measure the forward
scatter (FSC) from the objects.
Detectors perpendicular to the
beam measure side scatter (SSC)
and fluorescence.
13. • Forward scatter is based on two
properties: size and refractive
index
• The FSC intensity roughly
equates to the particle’s size and
can also be used to distinguish
between cellular debris and
living cells.
• Dead cells have lower FSC and
higher SSC than living cells.
14. • Side scatter is based on the
granularity or internal
complexity.
• The more granular the cell, the
more side scatter light is
generated.
15. • Fluorescence measurements taken at different wavelength can
provide quantitative and qualitative data about fluorochrome-labelled
cell surface receptors or intracellular molecules such as DNA and
cytokines.
• When a fluorescent dye is conjugated to a monoclonal antibody, it
can be used to identify a particular cell type based on the individual
antigenic surface markers of the cell.
• The stating pattern of each subpopulation, combined with FSC and
SSC data, can be used to identify which cells are present in a sample
and to count their relative percentages.
16. Optical detectors
• Scattered and emitted light from cells are converted to electrical pulses by
optical detectors.
• Once a cell or particle passes through the laser light, the scattered and
fluorescence signals are diverted to the detectors.
• Detectors are either silicon photodiodes or photomultiplier tubes (PMTs).
• The photodiode is less sensitive to light signals than the PMTs and thus is
used to detect the stronger FSC.
• PMTs are used to detect the weaker signals generated by SSC and
fluorescence.
19. Filters
• All the signals are routed to their
detectors via a system of filters and
dichroic mirrors.
• Each PMT fluorescence detector is
placed behind a series of dichroic
mirrors and filters, so that it only
receives and detect light within a
particular range of wavelength.
• A particular color of light is split off
from the incoming mixture and
directed to the detectors by dichroic
mirrors.
20. Types of filters:
• There are three major type of
filters:
1. Longpass Filter: Transmits
wavelength of light equal to or
greater than the spectral band
of the filter.
2. Shortpass Filter: Transmits
wavelength of light equal to or
shorter than the spectral band
of the filter.
3. Bandpass Filter: Transmits
wavelength of light within a
specific range of wavelengths.
LP 500 SP 500
Longpass
480 500 520
Shortpass
480 500 520
Bandpass
480 520
460 500 540
BP 500
21. 3.3 Electronics (Signal Processing)
• Scattered and emitted light data can
be converted to electrical pulses by
optical detectors.
• Flow cytometry data may be
represented as histograms or dot
plots.
22. Histogram: A histogram quantifies the
intensity of a single parameter, be it
fluorescence or scattering (SSC or FSC).
Dot plot: A dot plot is a two parameter
representation of a sample’s properties.
23. Gating
• Gating – It is a procedure to selectively visualize the cells of interest
while eliminating results from unwanted cells and debris.
• For example, if one has a heterogeneous population of cells which
contain lymphocytes, monocytes and granulocytes and is only
interested in evaluating the fluorescence of the lymphocytes
subpopulation.
• In this situation one could define an analysis gate around the
lymphocyte population. The resulting display would reflect the
fluorescence properties of only lymphocytes.
24. • For example, if we want to analyse a sample of peripheral blood that had been stained with
fluorochromes to identify the CD4 and CD14 surface markers but we are only interested in
knowing the percentage of monocytes that contain CD4 and CD14 surface markers. We will
place a gate around the monocyte population of the FSC versus SSC scatter plot.
26. 4.1 Cell Sorting (FCAS)
• A major application of flow
cytometry is the physical
separation of sub-population of
cells of interest from a
heterogeneous population.
• This process is known as cell
sorting of Fluorescence
Activated Cell Sorting (FACS)
New drop
Positive Charge
Negative Charge
No Charge
27. • Most commonly used cell sorting method is electrostatic deflection of droplets.
• In this method, the stream is focused in a vibrating nozzle and exits in a jet which
is broken into regularly spaced droplets.
• The cells of interest are charged electrically (positively or negatively). The
electrical charging actually occur at a precise moment called the ‘break-off point’.
• The cell of interest gets charged at the break-off point.
• When a charged droplet passes through a high voltage electrostatic field,
between the deflection plates, it is deflected and collected into the
corresponding collection tube.
• The deflection of the droplets is towards the oppositely charged plate, so that
this droplet is separated from uncharged and oppositely charged droplets.
29. The following features of the apoptotic cascade
can be observed using flow cytometry:
• Altered phospholipid composition in the plasma membrane
• Activation of caspases
• Chromation condensation
• DNA fragmentation
• Expression of proteins involved in apoptosis
• Changes in mitochondrial membrane potential
• Decrease cytosolic pH
• Altered membrane permeability
30. Detection of apoptotic cells based on changes
in forward scattering
• During apoptosis there is an initial increase in SSC (probably due to
the chromatin condensation) with a reduction in FSC (due to cell
shrinkage).
• Drawback: In many cases, the forward light scattering histograms of
apoptotic and live cells overlap and make it difficult to discriminate
apoptotic cells based solely on this parameter.
31. Detection of apoptotic cells based on Annexin
V binding
• During early apoptosis cell lose
symmetry, phosphatidylserine
on the outer leaflet of the
plasma membrane.
• Annexin V is a calcium-
dependent phospholipid-binding
protein that binds preferentially
to negatively-charged
phosphatidylserine.
32. • The assay involves incubating cells briefly in a solution containing
fluorochrome conjugated Annexin V (FITC – Annexin V) in a buffer
that facilitates its binding.
• Apoptotic cells can be detected easily by flow cytometry on the basis
of fluorescence due to increased binding of FITC-conjugated Annexin
V.
• Drawback: Unfortunately, it is not specific only for apoptosis because
whenever cell membrane integrity is disrupted (even non-ionic
detergents), cells may stain with Annexin V.
33. Detection of apoptotic cells based on PI
binding
• The intact plasma membrane of live cells have a tendency to exclude
cationic dyes such as propidium iodide (PI) and 7-amino-
actinomycin D (7-AMD).
• PI is a good staining method to distinguish apoptotic, necrotic and
normal live cells.
• Apoptotic cells show an uptake of PI that is much lower than that of
necrotic cells. It is therefore possible to distinguish live (PI-negative),
apoptotic (PI-dim) and necrotic (PI-bright).
34. • Thus, the combined use of cationic dyes (e.g. PI) with annexin V
allows the discrimination between:
Live cells = Annexin V negative/PI negative
Early apoptotic cells = Annexin V positive/PI negative
Late apoptotic = Annexin V positive/PI positive
Necrotic cells = Annexin V negative/PI positive
35. Detection of apoptotic cells based on DNA
Fragmentation
• The late stages of apoptosis are characterized by changes in nuclear
morphology, including DNA fragmentation, chromatin condensation,
degradation of nuclear envelope, nuclear blebbing and DNA strand
breaks.
• Cells undergoing apoptosis display an increase in nuclear chromatin
condensation. As the chromatin condenses, cell-permeable nucleic
acid stains become hyperfluorescent, thus enabling the identification
of apoptotic cells.
36. Assessment of mitochondrial membrane
potential and caspases level
• Assessment of mitochondrial membrane potential and caspases level
within the cell through flow cytometry is also used to analyse
apoptotic cells.
• Cells undergoing apoptosis often lose the electric potential that
normally exists across the inner mitochondrial membrane.
• A distinctive feature of early stages of apoptosis is the activation of
caspases enzymes. These enzymes can be labelled with fluorophore
which can easily be detected by flow cytometry.
37. 4.3 Cell Cycle Study
• One of the important application of flow cytometry is the
measurement of DNA content in cells.
• The duplication of the DNA occurs during the S-phase of cell cycle.
• There are two differnet methods to measure the DNA content:
1. The cells have to be stained with a fluorescent dye that binds DNA in a
stoichiometric manner.
2. Incorporation of thymidine analog bromodeoxyuridine (BrdU) during new
DNA synthesis.
38. Fluorescent dye that binds DNA in a
stoichiometric manner
• The cells are treated with a fluorescent dye that stains DNA
quantitatively. The fluorescence intensity of the stained cells at
certain wavelengths will therefore correlate with the amount of DNA
they contain.
• Dyes have different binding mechanism:
Intercalative binding dyes – Propidium iodide
A.T rich regions binding dyes – DAPI (4,6- Diamidion-2-phenylindole)
G.C rich regions binding dyes – Chromomycin A3
39. Incorporation of thymidine analog
bromodeoxyuridine (BrdU) during new DNA
synthesis
An accurate method for detection of
cell cycle progression also uses the
incorporation of thymidine analog
bromodeoxyuridine (BrdU) during
new DNA synthesis.
The incorporated BrdU is then stained
with specific fluorescently labelled
anti-BrdU antibodies, and the levels of
cell-associated BrdU measured using
flow cytometry.
By this method, the number of cells
that are proliferating rapidly & the
duration of S-phase can be calculated.
40. Clinical Applications of Flow Cytometry
1. Diagnosis of Hematologic
Malignancies
2. Detection of Minimal Residual
Disease
3. Lymphocyte Subset
Enumeration (HIV)
4. Efficacy of Cancer
Chemotherapy
5. Reticulocyte Enumeration
6. Cell Function Analysis
7. Application in Organ
Transplantation
After hydrodynamic focusing, each cell passes through a beam of light.
The number of detectors will vary according to the machine.
A particular color of light is split off from the incoming mixture and directed to the detectors by dichroic mirrors.
‘Y’ = Counts
‘X’ = Fluorescence Intensity
Subpopulations are defined as peaks in the histogram.
FITC – Fluorescein isothiocyanate dye
Light scatter plot
Fluorescence data from the gated region of monocytes population clarifies which cells contain surface surface markers (CD14 and CD4)
FITC – Fluoroscein isothiocyanate dye
PE – Phycoerythrin dye
Most commonly used cell sorting method is electrostatic deflection of droplets.
Apoptosis is a form of programmed cell death that occurs in multicellular organisms. Biochemical events lead to characteristic cell changes (morphology) and death.
PI = Propidium iodide
In addition to surface immunophenotyping and cytoplasmic characterization, flow cytometry is also used in cell cycle analysis.
Stoichiometric means the stain is directly proportional to the amount of DNA within the cell.