Introduction to Flow Cytometry IGC Workshop April 27-30, 2010 General Programme TimeTutorialRoomSpeaker Day 1 9h30-10h45Fundamentals of Flow CytometryIoniansRui Gardner (IGC) 11h00-13h00 Policies and procedures when using the IGC FACS equipment FACS roomTelma Lopes (IGC) Day 2 9h30-10h45Multicolor AnalysisIoniansRui Gardner (IGC) 11h00-13h00 Module 1 – Group 1 Module 2 – Group 2 FACS room Day 3 9h30-10h45Applications in Flow CytometryIoniansRui Gardner (IMM) 11h00-13h00 Module 1 – Group 2 Module 2 – Group 3 FACS room Day 4 9h30-10h45Cell SortingIoniansTelma Lopes (IGC) 11h00-13h00 Module 1 – Group 3 Module 2 – Group 1 FACS room Module 1 – Cell Cycle Analysis using FACScan (Telma Lopes) Module 2 – Multicolor Analysis using CyAn ADP (Rui Gardner)

What is Flow Cytometry? Flow Cytometry uic Fundamentals of Flow Cytometry IGC – April 27, 2010 Introduction to Flow Cytometry IGC Workshop

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

Resources Spectra Viewers: Tutorials: Books: Shapiro, H. “Practical Flow Cytometry”, 4ed, Online version. Purdue Univ. Cytometry Labs: all you need to know about Cytometry! 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

What is Flow Cytometry? 5 Cytometry: Measurement of physical and chemical properties of cells. Flow Cytometry: Characterization of cells flowing in a stream of fluid FACS: Fluorescence Activated Cell Sorting

Key Advantages of Flow Cytometry Population data Measure thousands of cells/particles per second Measure multiple parameters simultaneously. Detection and sorting of extremely rare populations Cell sorting High purity, speed, and yield. 0.02%

Flow Cytometers 7 FACSCalibur FACSscan FACSCanto LSR II CyAn ADP etc FACSAria MoFlo FACS Vantage EPICS ALTRA etc

The Instrument 8

Interrogation point 10

Hydrodynamic Focusing 11 More cells pass per unit time More cells pass per unit time Less cells pass per unit time Less cells pass per unit time Sheath Flow Sheath Flow Sample Flow Laser High Flow Rate Sheath Flow Sheath Flow Sample Flow Laser Low Flow Rate

Flow Rate 12 Low Flow Rate Laser Sheath Sample High Flow Rate Laser Sheath Sample

Lasers nm Most common laser wavelengths available

Light Scatter 16 Light is reflected towards all directions Low angle: Forward Scatter (FSC) High angle: Side Scatter (SSC)

Forward Scatter (FSC) 17 The magnitude of Forward Scatter is roughly proportional to the size of the cell.

Side Scatter (SSC) 18 Side Scatter is caused by granulosity and structural complexity inside the cell.

(2D) Scatter Plot 19

Fluorescence (1) 21 Low Energy High Energy Ground State Absorbed Light Excited State Emitted Light

Fluorescence (2) Low Energy High Energy

Excitation Spectrum (1) 23 Excited 480 nm520 nm550 nm590 nm

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

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

Excitation-Emission Spectrum (2) Emission maximaExcitation maxima

Fluorescence Charts

Fluorescence Spectra Viewers Invitrogen ( ) BD Biosciences( )

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 First dye-coupled antibody using fluorescein isothiocyanate (FITC) patented by Joseph Burckhalter and Robert Seiwald, in 1960 Direct methodIndirect method Pictures adapted from wikipedia

Tandem Dyes 31 PE-Alexa Fluor 700 PE-Cy5 PE-TxRed PerCP-Cy5.5 PE-Cy7 APC-Cy7

DNA and Cell Function Probes CalciumMembranepHCell TrackerDNAMitocondria Fura 2FM 1-43SNAFLECMRADAPINonyl Acridine O Indo 1FM 4-64SNARFCMTPXHOECHSTMitoSox Bapta/dyeDiOBCECFBMQCTOPRO3Mitotracker Calcium GreenANNINE 6pHRodoDMFDAPIRhodamine 123 Fluo 4Di4ANNEPS hqlysosensorCMTMRDRAQ5MitoProbe

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

Fluorescent Proteins (2) San Diego beach scene drawn with living bacteria expressing 8 different colors of fluorescent proteins

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