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Flow Cytometry Basic Training. What Is Flow Cytometry? Flow ~ cells in motion Cyto ~ cell Metry ~...
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Transcript of 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
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Models Models
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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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mitt
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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
ceT
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
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ittan
ceT
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mitt
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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
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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/