flowcytometry basics
TRANSCRIPT
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Flow Cytometry
Anna Merca - Philippe Melas - Tolga Sütlü
Stockholm Research School in Molecular Life Sciences
14 October 2005
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Outline
• IntroductionIntroduction
• How Does Flow Cytometry Work?How Does Flow Cytometry Work?
• ApplicationsApplications
• Focus on Analysing ApoptosisFocus on Analysing Apoptosis
• Cell SortingCell Sorting
• Benefits & LimitationsBenefits & Limitations
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Introduction
• Powerful analytical tool
• up to 10000 individual cells/sec
• many properties at the same time
• wide area of application
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How does it work?
• Cells in suspension (usually stained)
• 5 basic units– Flow cell – Light source– Optical filter units– Photomultiplier tubes– Data processing and operating unit (software)
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Direct beam stopLaser
Light
High angle scatter :(Side Scatter)Cell structure
Low angle scatter :(Forward Scatter)Cell size
Fluorescence at longerwavelengths
Flow cell
Direction of flow
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Basic Optics of a Flow Cytometer( An automated fluorescent microscope)
Dichroic mirrors 1 2 3
Laser(s)
Cell
CollectionLenses
ScatterLow & High
anglePhotomultiplier tubes
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Analysis Applications
• Viability and physiological state• Cell cycle analysis & nucleic acid content• Cell growth & death rates• Intracellular calcium concentration• Apoptosis• Biotechnological Applications
– Bacterial Cultivations– Yeast Cultivations– Mammalian Cell Cultivations
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Analyzing the graph-one color-
10 1 10 2 10 3 10 4
CD3 FITC -->
01
0
Num
ber
CD3
NUMBER
10 1 10 2 10 3 10 4
CD3 FITC -->
01
0
Num
ber
CD4
NUMBER
10 1 10 2 10 3 10 4
CD3 FITC -->
01
0
Num
ber
CD8
NUMBER
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Analyzing the graph
CD4
10 1 10 2 10 3 10 4
CD8 TC -->
10
11
02
10
31
04
Anti-
TC
R-g
am
ma-d
elta
-1 P
E -
->
10 1 10 2 10 3 10 4
CD3 FITC -->
01
0
Num
ber
0 1
10
21
03
10
4
010
CD3
10 1 10 2 10 3 10 4
CD8 TC -->
10
11
02
10
31
04
Ant
i-TC
R-g
amm
a-de
lta-1
PE
-->
CD3
CD4
CD3+CD4+ green
CD3-CD4+ cyan
CD3+CD4- cyan
CD3-CD4- black
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Apoptosis vs. Necrosis
• Suicide
• Genetically programmed
• Cells shrink
• Condensation of chromatin
• Internucleosomal degradation of DNA
• Membrane retains its integrity
• MMP reduced
• Murder
• Random Event
• Cells swell
• Non-specific loss of chromatin structure
• Non-specific degradation of DNA
• Membrane loses its integrity
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Apoptosis vs. Necrosis
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How to study apoptosis:
Caspases• Fact : Caspase 3 is activated in apoptosis
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How to study apoptosis: Mitochondrial Membrane Potential
• Fact: MMP is reduced during apoptosis
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How to study apoptosis: Plasma Membrane
• Fact: Phosphatidyl serine (PS) flips from inside to the outside of the membrane during apoptosis
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Cell Sorting 1
• sample nozzle is vibrated
• sample stream breaks up into regular droplets
• droplets are electrostatically charged prior to passing through the laser
• droplets containing cells of interest (as characterized by scatter or fluorescence properties) are deflected into tubes by passing them through a pair of charged plates
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Cell Sorting 2
ChargedPlates
Collection Tubes
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Benefits of Flow Cytometry
• measurement of single cells, identification of sub-populations
• wide area of application for analysis & diagnosis
• efficient and fast
• may be controlled remotely (online)
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Limitations
• can not tell the intracellular location and distribution of proteins
• aggregates or debris can give false results• pre-treatment of the cells for fluorescent
staining is time-consuming
• samples such as tissue or cells in culture have to be treated to separate cells
• expensive and needs highly-trained technicians
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Thanks for listening...