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* Flow = cells in motion * Cyto = cell * Metry = measure * Measuring properties of cells while in a fluid stream * Flow Sorting * Sorting (separating) cells based on properties measured in flow * Also called Fluorescence- Activated Cell Sorting (FACS) 4

What can flow cytometry be used for? Immunology Hematology Pathology Microbiology Genetics Drug discovery Toxicity testing Cell culture studies Functional studies Clinical and Research Chemical Engineering Biotechnology Animal Sciences 5

* It can be used for…  Immunophenotyping  DNA cell cycle  Membrane potential  Cell viability  Intracellular protein staining  Cell tracking and proliferation  Sorting  Chromatin structure  Total protein  Surface charge  Enzyme activity  Oxidative metabolism  DNA synthesis  DNA degradation  Gene expression * The use of flow in research has boomed since the mid-1980s 6

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Early instruments 10

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* Human Senses * Sight * Sound * Smell * Touch * Taste 12

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* Flow cytometry is a powerful tool for interrogating the phenotype and characteristics of cells. It is based upon the light-scattering properties of the cells being analyzed and these include fluorescence emissions. This fluorescence may be associated with dyes or conjugated to mAbs specific for molecules either on the surface or in the intracellular components of the cell 14

* Flow cytometry facilitates the identification of different cell types within a heterogeneous population * It was initially developed by immunologists wishing to separate out different cell populations for subsequent coculture experiments to determine the function of cells within the immune system * This was achieved by using fluorescence activated cell sorting, or FACS, on the flow cytometer. * The initial instruments were able to analyze one or two colors of fluorescence; today, instruments capable of analyzing 11 colors of fluorescence are available 15

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Flow Cytometer Instrumentation Graphical Summary 17

* All forms of cytometry depend on the basic laws of physics, including those of fluidics, optics, and electronics * Flow cytometry is a system for sensing cells or particles as they move in a liquid stream through a laser (light amplification by stimulated emission of radiation)/light beam past a sensing area 18

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* Size * Shape (Granularity & Density) * Makeup (Fluorescence Abs against markers) 20

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* Forward light scatter (FSC): proportional to cell size * Side light scatter (SSC): proportional to cell granularity * Fluorescence: * Binding of fluorescent-labeled antibodies * Ca ++ -sensitive dyes within cells * Fluorescent proteins expressed by cells * Binding of DNA dyes 22

* The amount of light scattered in the forward direction. * influenced by the size of cells. * can be used to distinguish live from dead cells 23

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detector Forward scatter, small angle scatter, FSC Diffracted light, related to particle’s surface area and refractive index, detected along axis of incident light in forward direction. Laser Sheath stream 25

* The amount of light scattered to the side is detected in the side or 90 o scatter channel * influenced by the granularity of cells * used to distinguish granulated cells from non-granulated cells 26

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90 Degree Light Scatter FSC SSC Laser 28

Side Scatter, Wide angle scatter, SSC Measure of cell granularity and complexity Refracted and reflected light Light scatters in all directions, but SSC usually measured 90 degrees from incident light 29

Sample stream Sheath stream Cell Laser beam Flow Cell 30

1. Cells in suspension flow single file past 2. Focused laser where they scatter light and emit fluorescence that is filtered and collected 3. Then converted to digitized values that are stored in a file Optic Fluidics Electronics Flow Cytometers are made of 31

* Need to have cells in suspension flow in single file * The cells from the sample tube are injected into the sheath fluid (PBS) * Flow in a flow cell is laminar. * Laminar flow: sample fluid flows in a central core that does not mix with the sheath fluid 32

Sample in Sheath Sheath in Laser beam Piezoelectric crystal oscillator Fluorescence Sensors Scatter Sensor Core Sheath Signal direction Flow Chamber 33

Sheath Tank Waste Tank SampleTube 34

* The flow chamber is instrumental in delivering the cells in suspension to the specific point that is intersected by the illuminating beam and the plane of focus of the optical assembly * Cells suspended in isotonic fluid are transported through the sensing system * To confine cells to the center of the flow stream; this also reduces blockage due to clumping 35

Sheath Sample Stream Cell The introduction of a large volume into a small volume in such a way that it becomes “focused” along an axis is called Hydrodynamic Focusing. 36

* LASER: Light amplification by stimulated emission of radiation * single wavelength of light (monochromatic) * coherent light (all emmiting photons have same wavelength, phase and direction) 37

Lasers Cytometers will have one or more lasers: Common excitation wavelengths: 488 (blue) 635 (red) 405 (violet) 532 (green) 350 (UV) 561 (yellow-green) 38

* Fluorochromes on/in the cell may absorb some of the light and become excited * As those fluorochromes leave their excited state, they release energy in the form of a photon with a specific wavelength, longer than the excitation wavelength 39

Flow Cell Injector Tip Focused laser beam Sheath fluid 40

* Many wavelengths of light will be scattered from a cell, we need a way to split the light into its specific wavelengths in order to detect them independently. This is done with filters * Optical filters : absorb or reflect some wavelengths of light, while transmitting other. 41

Filter placed at 45 o Reflected light Transmitted LightLight Source 42

Abdcerotec.com 43

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* Single Color Histogram * Fluorescence intensity (FI) versus count * Two Color Dot Plot * FI of parameter 1 versus FI of Parameter 2 * Two Color Density Plot * FI of P1 versus FI of P2. Areas of higher density will have a different color than other areas 47

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FSC 49

Positive Negative Count Fluorescence picked up from the FITC PMT 50

Mouse Lymph Node CD4 + T cell 51

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CD4 FITC Negative Population Single Positive Population Double Positive Population CD25 PE 54

 Is used to isolate a subset of cells on a plot  Allows the ability to look at parameters specific to only that subset 55

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Instrument Setup User adjusts sensitivity of detectors so that: – Events of interest are on scale – “Negative” fluorescence on the left/bottom, providing maximum dynamic range for positive signals 58

1- Setting Voltages Setting FSC and SSC Whole Blood Before optimizingOptimized 59

For each color, adjust voltage so that the negative population is in the first decade, off of the axis (if possible) 2- Setting Starting Voltages for Fluorescent Parameters 60

* Negative” signal on cells is auto fluorescence due to flavins, porphyrins and other molecules or properties of the material (plastics fluoresce in certain excitation wavelengths). * Different cells will have different levels of autofluorescence (e.g.lymphs vs. monos, different cell lines) affecting sensitivity in certain parameters with high base signals. 61

 Fluorochromes typically fluoresce over a large part of the spectrum (100nm or more)  Depending on filter arrangement, a detector may see some fluorescence from more than 1 fluorochrome  You need to “compensate” for this bleed over so that 1 detector reports signal from only 1 fluorochrome 62

 There is some overlap between the colors emitted by different fluorescent markers, therefore mathematical compensation is used to reduce overlapping results 63

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BD LSR II User’s Guide; Becton Dickinson 66

* The threshold can be set on any parameter, but is usually set on FSC 67

Forward scatter used as trigger signal. Events below cutoff are ignored. 68

Eliminates debris, RBC’s, platelets, instrument noise. Adjust FSC Threshold BeforeAfter 69

70 * Beckman Coulter * FC500, MCL-XL, Elite, Profile, Point Care * Becton Dickinson * Canto, FACSCalibur, FACSCan, FACSort, FACSCount * Guava Technologies Inc. * Personal Cell Analyzer System (PCA) * Partec - CyFlow

* Once the values for each parameter are in a list mode file, specialized software can graphically represent it. * The data can be displayed in 1, 2, or 3 dimensional format * Common programs include…  CellQuest  Flowjo  WinMDI  FCS Express 71

Side Scatter Forward Light Scatter Lymphocytes Monocytes Granulocytes largest and most granular population smallest and least granular population 72

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