Flow Cytometry Basic Training. What Is Flow Cytometry? Flow ~ cells in motion Cyto ~ cell Metry ~...

Flow Cytometry Basic TrainingFlow Cytometry Basic Training

What Is Flow Cytometry?What Is Flow Cytometry?

Flow ~ cells in motionCyto ~ cellMetry ~ measureMeasuring properties of cells while in a

fluid stream

Flow Cytometry applicationsFlow Cytometry applications It can be used for…

Immunophenotyping DNA cell cycle/tumor ploidy Membrane potential Ion flux Cell viability Intracellular protein staining pH changes Cell tracking and proliferation Sorting Redox state Chromatin structure Total protein Lipids Surface charge Membrane fusion/runover Enzyme activity Oxidative metabolism Sulfhydryl groups/glutathione 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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Year

Pu

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Models Models

Year

1976 FACS II

1991 FACS Vantage

1998 FACS Vantage SE

2000 FACS DiVa

1980 FACS IV/440

2003 FACS Aria

•Instruments model

Section IISection II

The 4 Main Components of a Flow Cytometer

What Happens in a Flow What Happens in a Flow Cytometer?Cytometer?

Cells in suspension flow single fileCells in suspension flow single file pastpasta focused laser where they scatter light and a focused laser where they scatter light and

emit fluorescence that is filtered and emit fluorescence that is filtered and collectedcollected

then converted to digitized values that are then converted to digitized values that are stored in a file stored in a file

Which can then be read by specialized Which can then be read by specialized software.software.

InterrogationInterrogation

FluidicsFluidics

ElectronicsElectronics

Interpretation

What Happens in a Flow What Happens in a Flow Cytometer (Simplified)Cytometer (Simplified)

cellflash.swf

The Flow CellThe 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 Hydrodynamic Focusing.Focusing.

Forward ScatterForward Scatter

FSCFSCDetectorDetector

Laser BeamLaser Beam

Side ScatterSide Scatter

FSCFSCDetectorDetector

CollectionCollectionLensLens

SSCSSCDetectorDetector

Laser BeamLaser Beam

Why Look at FSC v. SSCWhy Look at FSC v. SSC Since FSC ~ size and SSC ~ internal structure, a

correlated measurement between them can allow for differentiation of cell types in a heterogenous cell population

FSC

SS

C

Lymphocytes

Monocytes

Granulocytes

RBCs, Debris,RBCs, Debris,Dead CellsDead Cells

Fluorescence DetectorsFluorescence Detectors

FSCFSCDetectorDetector

CollectionCollectionLensLens

Laser BeamLaser Beam

FluorescenceFluorescenceDetector A, B, C, etc…Detector A, B, C, etc…

Spectra of Common Spectra of Common FluorochromesFluorochromes

Laser Lines (nm)Laser Lines (nm) 350350 457457 488488 514514 610610 632632

300300 400400 500500 600600 700700

PE-Texas Red

Texas Red

PI

Ethidium

PE

FITC

cis-Paranaric Acid

FiltersFilters

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 are designed such that they absorb or reflect some wavelengths of light, while transmitting other.

3 types of filters Long Pass filter Short Pass filter Band Pass filter

Long Pass FiltersLong Pass Filters

Transmit all wavelengths greater than specified wavelength Example: 500LP will transmit all wavelengths

greater than 500nm

400nm400nm 500nm500nm 600nm600nm 700nm700nm

Tra

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ittan

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Short Pass FilterShort Pass Filter

Transmits all wavelengths less than specified wavelength Example: 600SP will transmit all wavelengths

less than 600nm.

400nm400nm 500nm500nm 600nm600nm 700nm700nm

Tra

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ittan

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rans

mitt

ance

Band Pass FilterBand Pass Filter

Transmits a specific band of wavelengths Example: 550/20BP Filter will transmit

wavelengths of light between 540nm and 560nm (550/20 = 550+/-10, not 550+/-20)

400nm400nm 500nm500nm 600nm600nm 700nm700nm

Tra

nsm

ittan

ceT

rans

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Dichroic FiltersDichroic Filters

Can be a long pass or short pass filter Filter is placed at a 45º angle to the incident light Part of the light is reflected at 90º to the incident

light, and part of the light is transmitted and continues on.

DichroicDichroicFilterFilter

Detector 1Detector 1

Detector 2Detector 2

Example Channel LayoutExample Channel Layout

Dichroic Mirro

rs

Bandpass Filters

Detector#1Detector#1

Detector#2Detector#2

Detector#3Detector#3

Detector#4Detector#4

DetectorsDetectors

There are two main types of photo detectors used in flow cytometry PhotodiodesPhotodiodes

o Used for strong signals, when saturation is a potential problem (eg. FSC detector)

Photomultiplier tubes (PMT)Photomultiplier tubes (PMT)o More sensitive than a Photodiode, a PMT is used for

detecting small amounts of fluorescence emitted from fluorochromes.

Photoelectric EffectEinstein- Nobel Prize 1921

What is the puls?What is the puls?

cellflashelectronics.swf

InterpretationInterpretation

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

PlotsPlotsContour Plot Density Plot

Greyscale Density Dot Plot

GatingGating

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

specific to only that subset Can use boolean logic to include or exclude

multiple gates

Gating ExampleGating Example

Flow Cytometry Data

•Smaller Region, Live cells mostly

•Larger Region includes all cells

Red Detector

Blu

e D

etec

tor

PE detector

Red Detector

Blu

e D

etec

tor

PE detector

ReferencesReferences

Numerous References available in the Flow Lab Cytometry Current Protocols in Flow Cytometry Many more reference books available

Purdue University Cytometry Laboratories website: http://www.cyto.purdue.edu/ Dr. Robert Murphy, Carnegie Mellon University- Basic

Theory 1 and 2 powerpoint slides The Scripps Research Institute Flow Cytometry

Core Facility: http://facs.scripps.edu/