Flow Cytometry

Anna Merca - Philippe Melas - Tolga Sütlü

Stockholm Research School in Molecular Life Sciences
14 October 2005
 Outline
• Introduction
• How Does Flow Cytometry Work?
• Applications
• Focus on Analysing Apoptosis
• Cell Sorting
• Benefits & Limitations
 Introduction
• Powerful analytical tool
• up to 10000 individual cells/sec
• many properties at the same time
• wide area of application
 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)
 Flow cell High angle scatter :
(Side Scatter)
Cell structure
Direction of flow
Low angle scatter :
(Forward Scatter)
Cell size
Laser
Direct beam stop
Light

Fluorescence at longer
wavelengths
Laser(s) Basic Optics of a Flow Cytometer
( An automated fluorescent microscope)

Dichroic mirrors
1 2 3

Cell

Collection
Lenses

Scatter
Low & High
angle
Photomultiplier tubes
 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
 Analyzing the graph
-one color-
10

10
10
N N N
U U U
M M M
Number

Number
Number

B B B
E E E
R R R
0

0
0

10 1 10 2 10 3 10 4
10 1 10 2 10 3 10 4 10 1 10 2 10 3 10 4

CD3 FITC --> CD3 FITC --> CD3 FITC -->

CD3 CD4 CD8
 Analyzing the graph

4
10
10

Anti-TCR-gamma-delta-1 PE -->
3
10
C
Number

2
10
4

1
10
10 40

10 1 10 2 10 3 10 4

CD3 FITC -->

4
10
Anti-TCR-gamma-delta-1 PE -->
3
10

3
10

10 1 10 2 10 3 10 4

CD4 CD3
CD8 TC -->

CD3+CD4+ green
2

2
10

10

CD3-CD4+ cyan
1
0
1
10

CD3+CD4- cyan

10 1 10 2 10 3
10

10 4
0
CD3-CD4- black
CD8 TC -->
CD3
 Apoptosis vs. Necrosis
• Suicide • Murder
• Genetically programmed • Random Event
• Cells shrink • Cells swell
• Condensation of • Non-specific loss of
chromatin chromatin structure
• Internucleosomal • Non-specific degradation
degradation of DNA of DNA
• Membrane retains its • Membrane loses its
integrity integrity
• MMP reduced
Apoptosis vs. Necrosis
 How to study apoptosis:
Caspases
• Fact : Caspase 3 is activated in apoptosis
 How to study apoptosis:
Mitochondrial Membrane Potential
• Fact: MMP is reduced during apoptosis
 How to study apoptosis:
Plasma Membrane
• Fact: Phosphatidyl serine (PS) flips from inside to
the outside of the membrane during apoptosis
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
Cell Sorting 2

Charged
Plates

Collection
Tubes
 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)
 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
Thanks for
listening...