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*Flow CytometryBy: Dr. AkhtariAssistant Professor of Medical Nanotechnology, Department of Nanobiomedicine, School of Medicine, Mazandaran University of Medical Sciences

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* Principles of flow cytometry

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*What Is Flow Cytometry?

*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)

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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

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*Uses of Flow Cytometry

*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

Medline Publications citing "Flow Cytometry"

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*Brief history of flow cytometry

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Early instruments

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*Modern flowcytometrs

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*How do we measure things?

*Human Senses

*Sight

*Sound

*Smell

*Touch

*Taste

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*What is the difference with spectrofluorimetry?

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*summary

*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

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*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

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*It is too complicated!

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Flow Cytometer InstrumentationGraphical Summary

www.users.path.ox.ac.uk

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*Principles of Flow Cytometry

*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

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*What does flow cytometry measure

about cells?

*Size

*Shape (Granularity & Density)

*Makeup (Fluorescence Abs against markers)

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* What Happens in a Flow Cytometer (Simplified)

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*What measurements can be made?

*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

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*Forward Scatter Channel

*The amount of light scattered in the forward direction.

*influenced by the size of cells.

*can be used to distinguish live from dead cells

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detector

Forward scatter,small anglescatter, FSC

Diffracted light, related toparticle’s surface area andrefractive index, detectedalong axis of incident light inforward direction.

Laser

Sheath stream

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*Side Scatter Channel

*The amount of light scattered to the side is detected in the side or 90o scatter channel

*influenced by the granularity of cells

*used to distinguish granulated cells from non-granulated cells

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90 Degree Light Scatter

FSC

SSC

Laser

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Side Scatter, Wide angle scatter, SSCMeasure of cell granularity and complexity

Refracted and reflected light

Light scatters in all directions, but SSC usuallymeasured 90 degrees from incident light

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*Cytometer fluidics create laminar flow

Sample stream

Sheath stream

Cell

Laser beam

Flow Cell

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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

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*Fluidics

*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

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*Hydrodynamic SystemsSample in

Sheath

Sheath in

Laser beam

Piezoelectriccrystal oscillator

FluorescenceSensors

Scatter Sensor

Core

Sheath

Signaldirection

Flow Chamber

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*Fluidics Schematic

SheathTank

WasteTank

SampleTube

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*flow chamber

*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

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*The Flow Cell

Sheath

Sample StreamCell

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.

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*Lasers*LASER: Light amplification by stimulated emission of radiation

*single wavelength of light (monochromatic)

*coherent light (all emmiting photons have same wavelength, phase and direction)

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Lasers

Cytometers will have one or more lasers:

Common excitation wavelengths:

• 488 (blue)

• 635 (red)

• 405 (violet)

• 532 (green)

• 350 (UV)

• 561 (yellow-green)

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*Fluorescence Channels

*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

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Flow Cell

Injector Tip

Focused laserbeam

Sheath fluid

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*Filter Properties

*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.

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*Dichroic Filter/Mirror

Filter placed at 45o

Reflected light

Transmitted LightLight Source

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Abdcerotec.com

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*Different plots & gating

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*Types of Plots

*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

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*SSC/FSC

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FSC

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* 1 Parameter Histogram

1 2 3 4 6 7

150 160 170 .. 190

Positive

Negative

Count

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Fluorescence picked up from the FITC PMT

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*Single Color Histogram

Mouse Lymph NodeCD4+ T cell

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*2 Parameter

CD4 FITC

Negative Population

Single Positive Population

Single Positive Population

Double Positive Population

CD25 PE

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*Gating

Is used to isolate a subset of cells on a plotAllows the ability to look at parameters specific to only that subset

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*Instrument setting

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Instrument Setup

• User adjusts sensitivity of detectorsso that:

– Events of interest are on scale

– “Negative” fluorescence on theleft/bottom, providing maximumdynamic range for positive

signals

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1- Setting Voltages Setting FSC and SSC

WholeBlood

Before optimizing Optimized

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For each color, adjust voltage so that thenegative population is in the first decade,off of the axis (if possible)

2- Setting Starting Voltages for FluorescentParameters

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*Auto fluorescence

*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.

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*3- Compensation

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

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*Compensation

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

http://www.bdbiosciences.com

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66BD LSR II User’s Guide; Becton Dickinson

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*3- Threshold

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

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

69Eliminates debris, RBC’s, platelets, instrument noise.

Adjust FSC Threshold

Before After

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*Flow Cytometers

*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

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*Interpretation

*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…

CellQuestFlowjoWinMDIFCS Express

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*Scatter profile of lysed whole blood

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Forward Light Scatter0 200 400 600 800 1000

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Lymphocytes

Monocytes

Granulocytes

largest and most granular population

smallest and least granular population

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*Cell sorting

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