محاضرة من ضمن محضرات تكنولوجيا حيوية للأستاذ الدكتور محمد لبيب سالم لطلاب الفرقة الرابعة كلية العلوم تخصص علم الحيوان وكيمياء - جامعة طنطا

1. Principles of Flow CytometryPrinciples of Flow Cytometry
Mohamed Labib Salem, PhD
Prof. of Immunology
Founder and Executive Director
Center of Excellence in Cancer Research
Tanta University, Egypt

3. Cell
phenotype/genotype
– Cell numbers
– Cell size
– Cell subsets
– Cell apoptosis
– Cell necrosis
– Cell cycle
– Chromosome
– Cell sorting
Cell
function
• Cellular function
– Proliferation
– Activation
• Molecular features
– Protein secretion
– Metabolites
– Signaling pathways
– MHC/HLA
What do you need from your cells?What do you need from your cells?

4. How do you analyze your cells?How do you analyze your cells?
• Histology/HistopathologyHistology/Histopathology
• HistochemistryHistochemistry
• ImmunohistochemistryImmunohistochemistry
• BiochemistryBiochemistry
• Magnetic- activated cell sorting (MACS)Magnetic- activated cell sorting (MACS)
• ELISAELISA
• ELISASpotELISASpot
• Genetically:Genetically: FISH, PCR, DNA sequencing
• OmicsOmics
– Proteomic
– Genomic

5. Flow Cytometry: Is the technologyFlow Cytometry: Is the technology
QualitativeQualitative
QuantitativeQuantitative
Flow Cytometer: Is the equipmentFlow Cytometer: Is the equipment

6.  Flow ~ in motion
 Cyto ~ cell
 Metry ~ measure
• Measuring properties of single cells in a
fluid stream
• Gives us the ability to analyze many
properties of many cells in very little time
Flow Cytometry: Definition

7. Flow CytometryFlow Cytometry
• It is routinely used in the diagnosis ofIt is routinely used in the diagnosis of
health disorders, especiallyhealth disorders, especially blood cancersblood cancers,,
but has many other applications inbut has many other applications in basicbasic
research, clinical practice and clinicalresearch, clinical practice and clinical
trials.trials.
• A common variation is to physically sortA common variation is to physically sort
particles based on their properties, so asparticles based on their properties, so as
toto purify populations of interestpurify populations of interest..

8. Flow Cytometry:Flow Cytometry: DefinitionDefinition
• In biotechnology, flow cytometry is a laser-based,
biophysical technology employed in cell
counting, cell sorting, biomarker detection
and protein engineering, by suspending cells in a
stream of fluid and passing them by an electronic
detection apparatus.
• It allows simultaneous multiparametric analysis of
the physical and chemical characteristics of up to
thousands of particles per second.

9. Flow Cytometry:Flow Cytometry: HistoryHistory
• The first flow cytometry device was disclosed in U.S.
Patent 2,656,508, issued in 1953, to Wallace H. Coulter.
• Mack Fulwyler was the inventor of the today's flow
cytometers in 1965 with his publication in Science.
• The first fluorescence-based flow cytometry device (ICP
11) was developed in 1968 by Wolfgang Göhde from
the University of Münster and first commercialized in
1968/69 by German developer and manufacturer
PartecPartec.
• The first FACS (Fluorescence-activated cell sorting)
instrument from Becton Dickinson (1974), the ICP 22
(1975) from Partec/Phywe and the Epics
from Coulter (1977/78).

10. Parameter Microscopy Flow Cytometry
Means of Analysis Cells immobilized on microscope
slide, counted manually
Cells suspended in liquid stream,
pass single file in front of laser
Means of Detection Eye Electronic
Number of Cells Analyzed Hundreds Thousands-Millions
Rate of Analysis 100 cells per minute 2000-5000 cells/second
Sensitivity Low-only strong protein
expression can be detected
High-Weak protein expression
can be clearly identified.
Data Type Qualitative-Cells scored as +/- Quantitative-Fluorescence of
each cell individually scored
LASER
FLUORESCENCE
DETECTOR
FLUORESCENCE
DETECTOR
MICROSCOPY FLOW CYTOMETRY

11. Flow Cytometry: 5 Requirements
Single cell suspension
Antigen- antibody conjugate
Fluorochrome & Fluorescence
Flow Cytometer
Cell analysis software

12. Chromosomes
Nuclear membrane
Nucleus
Plasma membrane

13. Centrioles
Cytoskeleton Endoplasmic reticulum
Ribosomes

14. LysosomeChloroplasts
Mitochondria
Golgi apparatus

17. Research & Clinical applications of the instrumentBD FACSCanto Applications

18. Flow Cytometry: 5 Steps
Cell Preparation
Staining using labeled-Abs
Cell Acquisition
Cell Analysis
Data interpretation

19.  You need to have the cells flow one-
by-one into the cytometer to do
single cell analysis
Flow Cytometry: Cells

20. Flow Cytometry: Antigens
Antigens
• Serve as cell identifiers
• T-cells have surface antigens CD4 and CD8 on
their surface
• B-cells have surface antigens CD19, CD20, etc
Antigens
• Serve as cell identifiers
• T-cells have surface antigens CD4 and CD8 on
their surface
• B-cells have surface antigens CD19, CD20, etc
B cell
T cell
Cell surface
antigens

21. Flow Cytometry: Antibodies
ANTIBODIES:
 Proteins used
by the immune
system to
neutralize
foreign
invaders
 Recognize,
through
specific binding
antigens
ANTIBODIES:
 Proteins used
by the immune
system to
neutralize
foreign
invaders
 Recognize,
through
specific binding
antigens

22. Polyclonal antibodies
•Immunochemically dissimilar
antibodies
• React with various epitopes on a
given antigen
Monoclonal antibodies
• Immunochemically identical antibodies
• React with a specific epitope on a given
antigen
Flow Cytometry: Basis

23. Flow Cytometry: Labeling
ANTIBODIES:
 Labeling with
dyes
ANTIBODIES:
 Labeling with
dyes

24. Flow Cytometry: Labeling

28. Flow CytometryFlow Cytometry
How it worksHow it works

33. Flow Cytometer: Components

34. Flow Cytometry: ComponentsFlow Cytometer: Components

36. How it worksHow it works
https://www.youtube.com/watch?
v=TDRhCWaYRsg

37. Flow Cytometer: Types
BD FACSCalibur
Moxi Flow™
Partec Flow Cytometer
BD FACSVantageBD FACSAria

38. BD FACSCanto II cytometer
Flow Cytometer: Types

39. Analysis of
solid tumor
Analysis of
blood
malignancies
Stem cell
phenotype
numbers
Bacterial
analysis
Viral
analysis
Reticulocyte
analysis
Detection of
Immunodeficie
ncy diseases
Detection of
autoantibodies
BD FACSCanto ApplicationsBD FACSCanto ApplicationsBD FACSCanto ApplicationsBD FACSCanto Applications
Sperm
analysis
Fungal/para
sitic
analysis
Research & Clinical applications of the instrumentConclusion
Apoptosis
Proliferation
Cell cycle
Chromosome
Analysis

40. Mohamed Labib Salem, PhD
Prof. of Immunology
Founder and Executive Director
Center of Excellence in Cancer Research
Tanta University, Egypt