Basics of Flow cytometry by Prof. Mohamed Labib Salem
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The document outlines the principles and applications of flow cytometry, a biophysical technology used for cell counting, sorting, and biomarker detection, essential in diagnosing health disorders and conducting research. It includes historical developments, operational fundamentals, and different types of flow cytometers, detailing their significance in analyzing single cells based on various characteristics. Additionally, it describes the steps involved in flow cytometry, including cell preparation, staining, acquisition, and data interpretation.
Basics of Flow cytometry by Prof. Mohamed Labib Salem
- 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
- 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
- 28. Flow CytometryFlow Cytometry How it worksHow it works
- 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