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Introduction to Flow Cytometry

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Presentation on theme: "Introduction to Flow Cytometry"— Presentation transcript:

1 Introduction to Flow Cytometry
IGC Workshop General Programme Time Tutorial Room Speaker Day 1 9h30-10h45 Fundamentals of Flow Cytometry Ionians Rui Gardner 11h00-13h00 Module 1 – Group 1 Module 2 – Group 2 Analyzer room (Hands-on) Day 2 Multicolor Analysis Module 1 – Group 3 Module 2 – Group 1 Day 3 Applications in Flow Cytometry Claudia Bispo Module 1 – Group 2 Module 2 – Group 3 Day 4 9h30-11h00 Cell Sorting 11h15-13h00 FlowJo Basic Training Giordano Bruno Module 1 – Cell Cycle Analysis using FACScan (Cláudia Bispo) Module 2 – Multicolor Analysis using CyAn ADP (Rui Gardner)

2 Flow Cytometry What is Flow Cytometry? Introduction to Flow Cytometry
IGC Workshop Flow Cytometry uic Fundamentals of Flow Cytometry Rui Gardner IGC – April 2, 2013

3 Overview Definitions: What is flow cytometry and what does it measure?
Fluidics: Hydrodynamic focusing, Flow Rate... Optics: Light, fluorescence, emission and detection... Electronics: pulse, data acquisition... Analysis: Tips on Data handling 3

4 Resources Purdue Univ. Cytometry Labs: all you need to know about Cytometry! Books: Shapiro, H. “Practical Flow Cytometry”, 4ed, Online version. Tutorials: History of Flow Cytometry: Shapiro, H. (2007) “Cytometry and Cytometers: Development and Growth”, in Flow Cytometry with Plant Cells (Eds, J. Dolezel, J. Greilhuber, J. Suda), WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 4

5 Resources (Web Tools) BD Biosciences:

6 Resources (Web Tools) Life Technologies:

7 Resources (Web Tools) eBioscience:

8 Resources (Web Tools) Bioledgend:

9 What is Flow Cytometry used for?
Measurement of physical and chemical properties of cells. Flow Cytometry: Characterization of cells flowing in a stream of fluid What is Flow Cytometry used for? Flow cytometry can be used to count large numbers of cells or particles based on size, internal compexity, phenotype, cellular state, cell function, DNA content, gene expression, and to quantify these same cellular properties at a single-cell level. Since the introduction of the world's first commercial as well as the first fluorescence-based flow cytometer, the ICP 11, in , key flow cytometry technologies have been pioneered by Partec. These include doublet discrimination, piezo-based closed sorting, software compensation and mobile flow cytometry systems. As a result the company holds an extensive collection of national and international key patents related to flow cytometry and cell analysis. Flow Cytometry is not equivalent to FACS: Fluorescence Activated Cell Sorting 9

10 Key Advantages of Flow Cytometry
Population data Measure thousands of cells/particles per second Measure multiple parameters simultaneously. Detection of extremely rare populations Cell sorting High purity, speed, and yield. 0.02% . Flow Cytometry does not... Locate where a certain component is within a cell Measure distribution of cellular components Measure cell morphology

11 Flow Cytometer in a nutshell
an illuminated volume where they scatter light and emit fluorescence that is filtered, collected and… Optics converted to digital values that are processed and analyzed in a computer. Electronics Cells in suspension flow in single file through… Fluidics 11

12 Flow Cytometers FACSscan (BD) MoFlo Legacy (BC) FACSCalibur (BD)
Analyzers High Speed Cell Sorters FACSscan (BD) FACSCalibur (BD) CyAn ADP (BC) LSR Fortessa (BD) FACSCanto (BD) EC800 (Sony-iCyt) Sony Spectral Analyzer (Sony) CyFlow Cube (Partec) MacsQuant (Miltenyi Biotec) Guava (Merck-Millipore) Gallios (BC) Accuri C6 (BD) Attune (Life Technologies) etc MoFlo Legacy (BC) FACSAria (BD) MoFlo XDP (BC) MoFlo Astrios (BC) Influx (BD) FACSJazz (BD) FACS Vantage (BD) EPICS ALTRA (BC) Avalon (Propel Labs) Synergy sy3200 (sony-iCyt) SH800 (Sony) etc 12

13 Fluidics

14 Interrogation point 14

15 Simplified Schematics
Sheath Flow Sample Injected Laser Flow Chamber Sheath Flow Sample Injected Laser Flow Chamber Hydrodynamic Focusing Flow Chamber in a BD FACSAria Cell Sorter Flow Chamber in a BD LSR Fortessa

16 Flow Rate Less cells pass per unit time More cells pass per unit time
CyAn ADP Sheath Flow Sample Laser Low Flow Rate Sheath Flow Sample Laser High Flow Rate LSR Fortessa Less cells pass per unit time More cells pass per unit time FACScan FACSCalibur 16

17 Flow Rate Low Flow Rate High Flow Rate
Laser Sheath Sample High Flow Rate Laser Sheath Sample High Power Lower Power High Power Lower Power Samples should be run at the lowest flow rate (differential pressure) as possible. If increasing flow rate has: No impact on population distribution: Use high flow rate. Changes population distribution: Use low flow rate and increase cellular concentration. 17

18 Typical jet-in air cytometer
Exposure time Typical jet-in air cytometer Cell velocity is proportional to Sheath Pressure 𝐯= 2∆𝐏 𝜌 v ~ 5-30m/s Laser beam height ~ 5-20 µm 𝐯 = 2∆𝐏 𝜌 𝐹 Therefore exposure time ~ s Typically, in sorters, sheath pressure can be reduced to increase exposure time, and therefore sensitivity and resolution of acquisition. Sheath pressure in Analyzers is usually fixed. 18

19 Optics

20 Excitation - Lasers Most common laser wavelengths available 20 488 nm
442 Most common laser wavelengths available 20

21 Multiple Laser Systems
Spatially Separated Laser Beams Laser Intercepts in Flow Cell FACSAria III (BD Biosciences) Laboratory of Molecular Immunology Institute Molecular Genetics, Academy of Sciences, Czech Republic FACSAria III (BD Biosciences) Time delay between lasers must be determined for each instrument for a given sheath pressure. Changes in Sheath Pressure will impact measurements. 21

22 Emission - Light Scatter
Light is reflected towards all directions Low angle: Forward Scatter (FSC) High angle: Side Scatter (SSC) 22

23 Forward Scatter (FSC) The magnitude of Forward Scatter is roughly proportional to the size of the cell. 23

24 Side Scatter (SSC) Side Scatter is caused by granulosity and structural complexity inside the cell. 24

25 (2D) Scatter Plot 25

26 Optics (Fluorescence)

27 Fluorescence (1) Excited State Ground State Fluorophore Energy Levels
Absorbed Light Emitted Light High Energy Stokes Shift Absortion Emission Ground State Low Energy Fluorophore Energy Levels 27

28 Fluorescence (2) Fluorophore Stokes Shift Low Energy High Energy 1 2 3
Absortion 1 3 Emission Fluorophore Lower Wavelength Higher Frequency Higher Energy Higher Wavelength Lower Frequency Lower Energy

29 Excitation-Emission Spectrum (2)
Excitation maxima Emission maxima Each Fluorochrome has a specific Excitation and Emission Spectra

30 Excitation Spectrum (1)
Excited 480 nm 520 nm 550 nm 590 nm 30

31 Excitation-Emission Spectrum (1)
Excitation max 550 nm

32 Excitation - Emission Excitation at different wavelengths
decreases intensity of emission.

33 Fluorescence Charts

34 Fluorescence Spectra Viewers (Web Tools)
BD: Life Technologies: Cell-Analysis/Labeling-Chemistry/Fluorescence-SpectraViewer.html eBioscience: Bioledgend: UArizona: Evrogen: 34

35 Optics (Fluorescent Markers)

36 Reactive and Conjugated Probes
Cascade Blue Pacific Blue Pacific Orange Alexa Dyes Lucifer yellow NBD R-Phycoerythrin (PE) PE-Cy5 conjugates PE-Cy7 conjugates PE-Texas Red PerCP TruRed PerCP-Cy5.5 conjugate Fluorescein (FITC) BODIPY-FL TRITC X-Rhodamine XRITC Lissamine Rhodamine B Texas Red Allophycocyanin (APC) APC-Cy7 conjugates Etc… Immunohistochemistry Direct method Indirect method First dye-coupled antibody using fluorescein isothiocyanate (FITC) patented by Joseph Burckhalter and Robert Seiwald, in 1960 Pictures adapted from wikipedia

37 Tandem Dyes PE-Alexa Fluor 700 PE-Cy5 PE-TxRed PerCP-Cy5.5 PE-Cy7
APC-Cy7 37

38 DNA and Cell Function Probes
Calcium Membrane pH Cell Tracker DNA Mitocondria Fura 2 FM 1-43 SNAFLE CMRA DAPI Nonyl Acridine O Indo 1 FM 4-64 SNARF CMTPX HOECHST MitoSox Bapta/dye DiO BCECF BMQC TOPRO3 Mitotracker Calcium Green ANNINE 6 pHRodo DMFDA PI Rhodamine 123 Fluo 4 Di4ANNEPS hq lysosensor CMTMR DRAQ5 MitoProbe

39 Fluorescent Proteins (1)
GFP was first noted by Shimomura et al., in the jelly fish Aequoria Victoria, in 1962 and cloned 30 years later

40 Fluorescent Proteins (2)
San Diego beach scene drawn with living bacteria expressing 8 different colors of fluorescent proteins Tsien Lab Roger Tsien, Nobel Lectures, 2009 - PLATE - Beach.jpg

41 Flow Cytometry What is Flow Cytometry? Introduction to Flow Cytometry
IGC Workshop Flow Cytometry uic Coffee Break…!

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