1. No category

How to define flow cytometry ?
Méthodes d’étude de la cellule
 The possibility to measure individually and simultaneously several
MV426
physical and biological characteristics of a cell in a heterogeneous
solution. It allows the identification of subpopulations and the
Flow cytometry…
estimation of an average population
To measure (-metry) optical cells properties (cyto-) through a fluid
 High rate (5.104 cells/s) and high sensitivity (100 antigenic
(flow) in front of an excitatory laser beam…
factors/cell)
 The possibility of separating physically every cell analysed
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CyFlow® Cube - Partec
LSR Fortessa
Becton Dickinson
Historic
1934 Moldavan - Cellular numeration : capillary + photoelectrique captor
1949 Coulter - Count, size (conductance variability)
1953 Crosland -Taylor - Use laminar flow as describes by Reynolds in 1883
1965 Kamentsky - Analysis of cells constituents (2 parameters : DNA + Proteins)
Fulwyler - Cell sorting using electrostatic methods
Gallios
Beckman Coulter
Some examples of…
1969 Van Dilla - Laser excitation
Flow cytometers : analysis
1972 Herzenberg - Cells sorting (1st article et 1st cytometers commercialization)
1978 Schlossman - Monoclonal antibodies production
Reinhertz - New fluorochromes development
2004 Perfetto - Analyze of 19 parameters (17 fluorescent signals)
2006 Chattopadhyay - Immunophenotypage by quantum Dots
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EasyCyte8HT
Guava Technologies
MACSQuant
MiltenyiBiotec
MoFlo XDP
Beckman Coulter
C6
Accuri
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Flow cytometry principle
1) Fluidic system
Astrios
Beckman Coulter
Some examples of…
3) Electronic system
Presents Particles in front
of the laser beam
Allows the conversion from
the
different
luminous
signals to electronic signals
which can be stored and
processed by the computer
via an appropriate software
Flow cytometers: analysis and cell sorting
Influx
Becton Dickinson
FACSAria
Becton Dickinson
2) Optical system
Invitrogen
Composed of one (or more) monochromatic
excitation source(s) (LASER), optical filters and
mirrors which select and separate emission
wavelength towards the appropriate detectors
FACSJazz
Becton Dickinson
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Fluidic system
Sample under pressure
Cells suspension
Sample pressure
hydrofocalization
Laminar flow
Psa low
sheath fluid
under pressure
Sample under pressure
Cells suspension
Carry away
Psa high
sheath fluid under
pressure
Sheath fluid flow induces a fast and
regular acceleration to cells and
forces them to be aligned in order
to be analyzed one by one
Flow Cell
Nozzel
Flow cell
Light excitation
Light excitation
Psa
Cell flow
 Separation
Carry vein
Weak focalization of cells
Tiny distribution
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Ps
Important pressure difference
Fast speed of passage
Weak pressure difference
Slow speed of passage
Important focalization of cells
 Alignment
Light excitation
(laser(s), lamp)
Psa
Ps
Large distribution
Psa = pression sample
Ps = pression sheath
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Optical system
Optical system
The cytometer records the behaviour of the cell trough the laser
beam, by measuring :
 Fluorescence emission (« Fluorescence Light » (FL))
Spontaneous (autofluorescence)
Associated to free or bound fluorochromes
 Light scattering (informs on the morphology and cell structure)
Detection at 90° granularity:
SSC or SS or 90LS
 Fluorescence are emitted in all directions and are always in another
color than the excitation laser
Detection at small angles
size : FSC or FS or FALS
Light excitation
Invitrogen
Invitrogen
- Light scattering at small angles (Forward Scattered Light, FSC) represents the light
incidence angles from the LASER and is directly proportional to the particle size
and area (relative measure of size)
- Light scattering at large angles (Side Scattered Light, SSC) measures the reflected
light and depends on granularity and complexity of the particle (relative measure of
 Color depends of the fluorescent probe nature
Invitrogen
 Induced fluorescence from different fluorochromes is separated by an
optical filters plot. The selection filters depends on the Excitation and
emission wavelengths
granularity)
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Optical system
Optical filter
 Filters and dichroic mirrors are part of optic elements. They realize the
separation of channels and select wavelength
 FSC signals are received by a photodiode. SSC signals and fluorescents
emissions are collected and diverted to photomultipliers tubes (PMT). Each
PMT have an optic filter which allow to detect specific wavelength
 Each fluorochrome is detected in a unique fluorescence optic channel:
emitted light follows a specific path from the “interrogation point” to the
detector
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 Optical
filters are materials which absorb
specific wavelength and transmit others
Incidence ray
Transmitted light
Absorbe light
 When filter and incidence light make a
45° angle the not absorbed wavelengths are
reflected  Dichroic mirrors
Reflected light
Transmitted light
Incidence ray
45°
Reflection
Transmission
Absorption
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Optical filter
Optical filter
 Different filters types used in CMF
 Different filters types used in CMF
525
500
475
l <475
l <500
 Transitions between absorption and transmission are not perfect
LP filter
100
100
50
0
wavelength l
50
0
100
50
0
50
0
SP 520
LP 520
BP 500
BP
500/40
500 +- 20
100
50
0
Band Pass filter
100
 example
BP filter
% de transmission
% de transmission
% of transmission
SP filter
100
% de transmission
640
570
l >500
Long pass filter
Short Pass filter
% de transmission
l >525
500/50
LP500
l <500
480
460
Band Pass filter
% of transmission
Long pass filter
SP500
l >500
Short Pass filter
wavelength l
480
50
440
0
wavelength l
480
520
560
wavelength l
600
640
440
480
520
560
600
wavelength l
640
440
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20
480
20
520
520
560
600
wavelength l
640
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Optical filter
Optical system
Band Pass 525/40
 Different filter types used in CMF
505
BP
525/40
525 +- 20
nm
545
Ex : Optical bench
Ligth sources
Light source
20 20
460
500
Dichroïc Long Pass 550
540
580
wavelength l
620
680
BD LSR cell analyzers
Invitrogen
Light source
460
500
540
580
wavelength l
620
680
BC Gallios
BC MoFlo XDP
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Optical system
Electronic system
 In order to measure the optical signals they have to be converted in
electric signals (volts).
 The choice of the detector depends on the photon number
Impulse formation
Flow
10
Laser
Diodes 1v  1e-
Volt
Laser
Volt
Detect intense signals (ex : size parameter)
Photomultipliers Tubes (PMT) 1v  ne-
Area (A)
Height (H)
Photons detectors
 The electrical signals created by light emission are called impulsions.
Their amplitude values lies between 0 and 10 volts
0 Width (W)
High sensitivity (ex : structural and fluorescent
parameters detector) used for weak signals
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Time (µ Seconds)
Laser
Time
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Electronic system
Electronic system
 electrical impulsion (volt) has to be transformed into numerical data by a
digital analogical converter (ADC)
1024
0
0
10
1024
0
0
10
1024
0
0
10
1024
0
0
10
1024
0
0
2
450
1024
 ex : An ADC of 10 bits
(210) enables 1024 values
Conversion
Light
excitation
3
4
 Linear
scale
Time
0
0
Photomultiplier
or
Logarithmic
5
Amplification
Flow
450
900
450
450
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Relative light
intensity
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Data acquisition
Data representation
 The cytometer stores all parameters of every cell : List mode (LMD)
SSC
FL1
FL2
FL3
Time
(µsec)
1rst
119
2de
124
65
541
797
669
1
86
120
842
669
3rd
1
223
252
574
837
730
2
4th
144
71
69
807
686
1
....
….
….
….
….
….
….
Last
112
87
574
83
655
1
UR
LL
LR
+
 Other two parameters representations
Cells number
UL
-
Relative light intensity
 Three-dimensional plot
Advantage : everything could be recalculated
842
 Two parameters histogram
 Histogram
Cells number
FSC
900
Arbitrary units
etc
 Each mesurement from each detector is called to as a parameter
Cells
analysis
900
Cells number
10
10
1
 Convert
into
binary
signal by the ADC, signals
are processed individually
arbitrary
units
Intensity
(volts)
Cells
83
120
574
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Threshold and trigger parameters
Data analysis
 Trigger parameters
 Regions permit to
Electronic treatment of the signal
Isolate groups of interest,
 Threshold
Often done on the size, it allows to reduce the number of events without interest
to not saturate the system
Measurement of amplification
0
10
Time (microsec.)
Volt
10
Monocytes
SSC
Threshold level
FSC
Lymphocytes
0
10
Time (microsec.)
SSC
Measurement of amplification
Threshold level
0
statistic calculations
Granulocytes
Volt
10
0
better discrimination by coloration,
FSC
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Data analysis
Data analysis
 Gates
 A few examples
Permit to combine logical regions, to define subgroups and to restrict the analyze
on interesting cells signals to characterize them (statistics)
Percentage
R1 : 68,94%
G2/M
R2 : 30,96%
R2
240
overlay
R3
R3 : 0,05
10 3
events
Counts
R2 mean : 333,75
R2
R2 : 1,29
10 4
FL2-Log_Height Comp
360 R1
R1
R1
R2
R1 mean : 6,45
Ungated
G0/G1
10 5
481
10 2
120
S
10 1
Gated
Doublets
0
100
2C
R5
R5 : 0,72
R4
R4 : 97,93
R1 and R2
101
102
103
FL1-Log_Height
104
105
10 0
100
101
102
103
FL1-Log_Height Comp
104
105
4C
Booleans equations
…etc.
AND
OR
NAND
NOR
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Doublet elimination
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Signal diffraction, refraction and reflection
 Refraction (dead or alive)
Laminar flow
 Diffraction (size)
Laser
G2 or M Doublet G1
2 x G1
events
G1
4n
Temps
1
Area
1
2
2
Height
Peak
1
2
1
(relative value)
1
Refraction is principally between 2 and 12°
angles: FSC
 Reflection (cell surface, nucleus,
granules…)
2n
Integral
Area
(relative value)
G2
2
Peak
Intensity Fluorescence
Excitation light
G1
Reflected light
Propidium Iodide
2
Refracted light
Excitation
light
Diffraction is principally between 0
and - 4° angles: FSC
Excitation light
Reflected light
Reflection is principally at 90° : SSC
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Physical properties
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Discrimination between living and dead cells
Deaths Cells
Granulocytes
Homogeneous cell size
Living cells
Forward scatter
Heterogeneous cell size
Monocytes
Forward scatter
Lymphocytes
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Fluorochrome characteristics
Brightness fluorochromes
Fluorochrome
Molecule induces an electronic transition which then induces fluorescence.
Associated to a macromolecule, it is called fluorophore
 Extinct coefficient multiplied by quantum output
 Excitation spectrum are compatible with the light source
ABS
EM
CD8-PerCP
wavelegnth (l)
 Quantum output 
CD8-FITC
CD8-ECD
CD8-APC
Ratio between the number of emitted photons and the number of absorbed photons
 Extinct coefficient e
Measurement of a reference fluorophore at a wavelength at 1M in a 1cm tank. (the more the
value the more the fluorescence is elevated)
 Signal stability
CD8-PC5
CD8-PC7
 Signal stochiometry
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CD8-PE
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Choice of fluorochromes
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Spectral overlapping
 …BUT for multicolor is very difficult…
 The excitation spectrum are compatible with your system
 Use brightest fluorochromes for least expressed proteins
 Use dimmest fluorochromes for most highly expressed proteins
 Choose association of fluorochromes to avoid overlaps...
 …hence limit spectral overlaps between fluorochromes and…
 calculate compensation…
two methods for compensation :
Medians method (manual)
Slope method (automatic with new generation system)
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Spectral overlapping
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Medians method
(Manual compensation)
Intensity
 A contamination percentage of a fluorochrome by another
B
FL1
FL2
530/40
580/30
 Tubes must contain negatives cells and monomarked cells (or beads)
 Photomultipliers voltage has to be high enough, allowing to have
negatives cells in first decade
 Gate must include positives and negatives
autofluorescence (ex gate on lymphocytes).
A
cells
of
a
same
 Adjust the compensation so as to positives and negatives cells, have
same median
 The calculated shift must be then entered in the system
This procedure must be done on each fluorophore (do not forget to
reset all parameters
Photomultiplier FL2 detects FITC fluorescence (A)
Photomultiplier FL1 detects PE fluorescence (B)
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Compensation – Medians method
Compensation
 A label (not a color) is attributed to each detector.
 The compensation method consists in removing a percentage from a
signal to another signal
10 5
Fluo1 Fluo2
Counts
10 2
Fluo2 -PE
736
402
552
301
Counts
PE - FL2-Log_Height
10 3
368
Compensation
etc.
PMT
ADC
201
184
100
10 1
R4
10 0
100
10 5
Fluo1 Fluo2
101
102
103
104
FITC - FL1-Log_Height
101
102
103
104
FITC - FL1-Log_Height
0
100
105
101
102
103
104
PE - FL2-Log_Height
105
105
 digital signal
R3
R2
Fluo1 -FITC
10 4
FL2-Log_Height Comp
PE - FL2-Log_Height
580/30
10 3
Counts
530/40
0
100
R5
10 2
Fluo2 -PE
736
402
165
552
301
123
Counts
Counts
Wavelength
Intensity
Fluo1 -FITC
10 4
580/30
Intensity
530/40
 Analog signal
R3
R2
368
PMT
201
82
Compensation
etc.
ADC
100
41
184
10 1
R4
10 0
Wavelength
100
0
100
R5
101
102
103
104
FITC
- FL1-Log_Height
FL1-Log_Height
Comp
105
101
102
103
104
FITC - FL1-Log_Height
105
0
100
101
102
103
104
PE - FL2-Log_Height
FL2-Log_Height
Comp
105
Compensation PE-%FITC
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Compensation
Log 4 decades
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From C. Aït Mansour - Miltenyi
Compensation
Log 5 decades
hyperLog
 Make compensation settings with bright fluorophores
 Do not touch values of PMTs after compensation settings
No compensation
Compensation
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From C. Aït Mansour - Miltenyi
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Required controls
Dilution of Antibodies
 Required controls
 Titrate antibodies
Example 5 colors
N°
Tube
FITC
PE
PE-TR
PE-CY5
PE-CY7
1
expérimental
CD3
CCR7
CD45 RO
CD27
CD8
2
non coloré
-
-
-
-
-
3
Ctrl isotypique
t
t
t
t
t
4
monomarquage
CD3
-/t
-/t
-/t
-/t
5
monomarquage
-/t
CCR7
-/t
-/t
-/t
6
monomarquage
-/t
-/t
CD45 RO
-/t
-/t
7
monomarquage
-/t
-/t
-/t
CD27
-/t
8
monomarquage
-/t
-/t
-/t
-/t
CD8
Secondary Ab 1/100
Secondary Ab 1/50
 Control with the same isotype and the
same dilution as the specific antibody
Primary Ab
1/25
1/50
1/100
 Useful controls : FMO (Fluorescence minus one)
9
Ctrl régions
-
CCR7
CD45 RO
CD27
CD8
10
Ctrl régions
CD3
-
CD45 RO
CD27
CD8
11
Ctrl régions
CD3
CCR7
-
CD27
CD8
12
Ctrl régions
CD3
CCR7
CD45 RO
-
CD8
13
Ctrl régions
CD3
CCR7
CD45 RO
CD27
-
1/200
Cytométry 69A : 1037-1042 (2006)
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Spreading data
Unstimulated control
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Cytométry 69A : 1037-1042 (2006)
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Antigens/ receptors
Excitation
Emission
Nucleic acids
Excitation
Emission
AMCA
354
442
DAPI, Hoechst 33258
354
442
A-T bases
Pacific blue
410
455
Hoechst 33342
354
463
A-T bases, vital
FITC
495
520
Phycoérythrine
495, 532, 560
575
PE-Rouge Texas*
495
620
PE-Cyanine 5°
495
670
Allophycocyanine
650
PE-Cyanine 5.5
488, 532, 560
PE-Cyanine 7
495
765
APC-Cy7
650
785
Sytox blue
430
480
Intercalate, viability
Cell trace calcein violet AM
405
450
hydrophobic
Red 613, ECD
YO-YO
470
510
ADN
Tricolor, quantum red. Visible light sensibility
Acridine Orange
500
530, 640
ADN/ ARN
660
TO-TO
510
533
ADN
720
Chromomycine
430
570
G-C bases
Sensible to pH
Pyronine Y
540
570
ARN
Propidium Iodure
536 (360 et 488)
617
Intercalate : ADN and/or ARN, viability
EMA, ethydium monoazide
510
600
Viability, covalent/ photo-achievable
7 A-actinomycine D
478
660
G-C bases, viability
CY7 could be altered by the fixation
Insensible to pH, soluble in water
Alexa : 350, 405, 430, 488, 532, 568, 594, 610, 633, 647, 660, 680, 700, 750
°PE/CY5 fixed monocytes and B lymphocytes of mice SJL, AKR, NOD
Rappel PE-CY5 excited at 488 nm by the emit energy transferred from PE but also by the laser 633
nm via CY5, compensations could be elevated or difficult to realize so try to replace PE-CY5 by PECY5.5 or PE-CY7 in a combination with APC
Amine reactive dyes : an effective tool to discriminate live and dead cells in polychromatic flow cytometry.
Perfetto SP, Chattopadhyay PK, Lamoreaux L, Nguyen R, Ambrozak D, Koup RA, Roderer M,.
J Immunol Metghods. 2006 Jun 30;313(1-2):199-208
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Cellular functions
Excitation
Monochlorobimane
360
420
glutathion
INDO-1-AM
335
485/ 410
Free/ bound Ca2+
FLUO-3
506
530
Free non fluo/ bound Ca2+ fluo
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Emission
Fura Red
480
660
Free fluo/ bond CA2+ non fluo
DIOC4 à 6, JC-1, CMXRos Rh 123
484
510
510
530
Mitochondrial potential
NAO (nonyl acridine orange)
489
525
BCECF
500
520/ 620
Mitochondrial mass membrane
SNARF
518
575/ 670
pH6/ 7,5
CFSE
495
530
pH6/ 9
FDG
490
520
Cellular proliferation
490
513
B-galactosidase
Apoptosis
Excitation
Emission
Phiphilux
488
530
Caspases-3
Annexin-V FITC
495
520
Phosphatidyl-serine
dUTP-FITC
495
520
3’-OH DNA ends
YO-PRO
495
520
ADN
SYTO-17
490
675
ADN
ECFP
433
475
EGFP
480
505
EYFP
513
527
490, 550
590
mRFP
580
610
 Spectrum excitation
U.V : Hoechst, DAPI
488 : IP, 7-AAD
Fluoresceine Diacetate FDA
DsRed
Ex : Nucleic acid dies
633 : TO-PRO3
 Permeant
Hoechst
Vybrant DyeCycle Violet stain
 Specificity
Non : IP
A-T : Hoechst
C-G : 7-AAD, Chromomicyne
 Toxicity
Hoechst
DRAQ5
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Cell Flow Sorting
Continuous jet sorting : principle
Partec System
Becton System
 Cell Flow sorting
Fluid
Separate cells in a flux according to their properties
Piezo-electric valve
 Fluorescence-Activated Cell sorting
FACS is a trademark of Becton Dickinson
Cells path when
opened valve
(unsorted cells)
Collection tube
Cells path when
closed valve (sort)
Center collection tube : sort
Off center collection tube : unsorted
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Cf C. Jayat Atelier INSERM 103
Sort “jet-in-air”
Electrostatic sorting
nozzle
 Droplet formation
1- Adjust sample and liquid sheath pressure.
2- Induce an amplitude variation and a variable frequency on the flow
stroboscope
 Drop delay
Time needed for one cell to go from the analyze point to the droplet
formation
 Target cell identification
Deviation plates
To analyze cells and to define sorting windows
Deflected jets
 Droplet charge
The drop charge determines the applicable tension on a droplet. Charged
droplet will be deviated from the normal path
 Droplet deviation
Waste bin
An electrostatic field deviate the droplet
Service Imagerie Cellulaire et Cytométrie en Flux
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A. Munier-01/2013
A. Munier-01/2013
Cell sorting “jet-in-air” : principle
Sort strategyB
A
Electric impulsion
 Variable position of the cell in the flow (coincidence notion)
nozzle
Time 0
Laser beam
Detector
Time 1
Drop Delay
Intersection
flux / laser
Time 0
Time 1
?
Last attached
droplet (DGA)
 A sorting envelop is defined depending on the direction needed for cell sorting
Deflection charged plate
+
+
+
Purity
1 droplet
Detection of coincidence ON
Purity/ Output
2 droplet
Detection of coincidence ON
Output/ Purity
3 droplet
Detection of coincidence ON
Output
3 droplet
Detection of coincidence OFF
-
Waste bin
2
Collecting tubes support
Service Imagerie Cellulaire et Cytométrie en Flux
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3
1
Service Imagerie Cellulaire et Cytométrie en Flux
A. Munier-01/2013
Application field
Studies
Morphological analysis size, numeration, cellular viability,
Antigenic analysis (membrane and cytoplasme)
phenotype of blood cells, hematology, leukemia's, antigenic
quantification, allergologic tests, prenatal diagnosis, specificity and
antibody titration...
 Fundamental and applicative research
 Medicine (hematology, immunology, cancerology …etc),
 Industry: food processing, environment, cosmetology, pharmacology
Functional analysis cellular signalization, metabolic activity, membrane integrity,
proteins and genetic expression, ionic flux, intracellular PH...
Nucleic acid analysis
DNA and RNA content, cellular cycle, proliferation, apoptosis, cellular
viability...
and toxicology.
Cell sorting
physiological criterions (fluorochromes), fluorescents telltale genes
(GFP…), phases of the cellular cycle, males or female spermatozoids...
Service Imagerie Cellulaire et Cytométrie en Flux
Service Imagerie Cellulaire et Cytométrie en Flux
A. Munier-01/2013
A. Munier-01/2013
Flow cytometry and safety
DNA/ cell cycle
Biological and chemical risks
G1
Phase G0/G1
21,8%
G2+M
33,6%
Linear markers give an
indication but it is just an
estimation
Cell Number
Electrical risks
44,6%
S
LASER risks
Phase S
Phase G2 + M
G1
41,6%
S
28,8%
G2+M
29,6%
Aerosols risks
Propidium Iodure
Mathematic models
without being perfect
Sound risks
are
more
precise
MultiCycle
Service Imagerie Cellulaire et Cytométrie en Flux
Service Imagerie Cellulaire et Cytométrie en Flux
A. Munier-01/2013
A. Munier-01/2013
Essential conditions for DNA content analysis
Cell cycle
Identification of mitosis cells
Apparition of the CyclineA2 during the cycle
IgG1
 Optimal use of the cytometer (marble of calibration, DNA beginning of
the fluorescence, correct voltage)
Cyclin A2 FITC
 Passing time : 200 events per second
 Coefficient of variation peak G0G1 < 5%
 Weak fragment proportion (<20%)
Ctrl
 Eliminate aggregates
Propidium Iodide
 10 000 cells minimum to calculate a cycle
S
G2/M
 Have diploid cells as reference (ex : chicken red blood cells…)
Propidium Iodide
MPM2 Alexa 488 fluorescence
 Utilization of a valid staining method for DNA
Cell Number
G0/G1
M
G2
DNA content PI fluorescence
J. Sobczak UMR 7622
Service Imagerie Cellulaire et Cytométrie en Flux
A. Munier-01/2013
Service Imagerie Cellulaire et Cytométrie en Flux
I. Gasnereau UMR 7622
A. Munier-01/2013
Modulation of the cell cycle
Cell cycle
Divers inhibitors effects of topo-isomerases
(treatment during 16 hours)
DNA peak
a tumorous cell line treated by antimitotic agent
% of cells in G2:
R1
R1
Cell number
R1
Reference: 16%
R1
2c
drug 0.1: 26%
drug 0.3: 50%
drug 0.9: 83%
1c
Asynchrones cells
Camptothecine
10 mM
Etoposide
250 nM
4c
ICRF-193
10 mM
Service Imagerie Cellulaire et Cytométrie en Flux
J. Sobczak UMR 7622
A. Munier-01/2013
Service Imagerie Cellulaire et Cytométrie en Flux
Agnès Chassevent - 2009
Paul Papin Center - Angers
Cell proliferation
A. Munier-01/2013
Fluorescence Ubiquitination Cell Cycle Indicator
(FUCCI)
Click-it EdU follows a protocol of
Aldehyde fixation and detergent permeabilization
Phase S
G2 + M
G1 + G0
0
• Fix for 15 minutes, wash
• Permeabilize for 30 minutes, wash
• Incubate in click labeling cocktail for 30 minutes, wash
• Optimal : incubate with cell cycle stain for 15-30 minutes
• Analyze
200
400
600
Control cells : EdU cells
800
EdU-incorporated
1000
FL3-H
Service Imagerie Cellulaire et Cytométrie en Flux
J. Sobczak UMR 7622
Service Imagerie Cellulaire et Cytométrie en Flux
A. Munier-01/2013
A. Munier-01/2013
Analysis of cell division
Clinical study of MDR*
*MDR Resistance MultiDrogue
Proliferation of T lymphocytes after 4 days of stimulation
D4
N°1
N°2
N°3
D6
events
D0
with verapamil
(carboxyfluoresceine diacetate, succimidyl ester)
CD56
CFSE
without verapamil
Rhodamine 123
A. Saoudi
Service Imagerie Cellulaire et Cytométrie en Flux
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Drénou, B
Service Imagerie Cellulaire et Cytométrie en Flux
A. Munier-01/2013
Effect of transfected gene on cell cycle
Ki-67 /DNA
200
transfected gene
150
32% of cells Ki-67
+
38%
# Cells
4
10
100
6.35
0
200
400 600
FL4-H
800
Ki-67 FITC
GFP
FL1-H
0
2
1000
Hoechst 33342
10
5000
1
10
93.2
Ki-67 FITC
50
3
10
# Cells
4000
3000
0
10
0
200
400
600
FL4-H
Hoechst 33342
800
1000
2000
20%
1000
Propidium Iodide (DNA)
0
0
200
400
600
800
1000
Service Imagerie Cellulaire et Cytométrie en Flux
Derek Davies, Cancer Research UK
A. Munier-01/2013
Propidium Iodide (DNA)
Service Imagerie Cellulaire et Cytométrie en Flux
Agnès Chassevent - 2009
Paul Papin Center – Angers
A. Munier-01/2013
Apoptotic event
Apoptotic event
Exposure of phosphatidylserine
to external face cytoplasmic membrane
intracellulare Ca++ increase
Indo1 emits blue fluorescence when it is free
Indo 1 emits purple fluorescence when it is binding Ca++
They measure ratio indo bind/indo free according to time
Propidum iodide
T human lymphocytes
non stimulated
T human lymphocytes
antigen timulated
Annexin V- FITC
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A. Munier-01/2013
Service Imagerie Cellulaire et Cytométrie en Flux
S. Valitutti
A. Munier-01/2013
Apoptotic event
DNA Fragmentation
TUNEL
pic sub-G1
(TdT-mediated X-dUTP nick end labelling)
2n
4n
Service Imagerie Cellulaire et Cytométrie en Flux
A. Munier-01/2013
J.F. Mayol – CRSSA - Grenoble
Service Imagerie Cellulaire et Cytométrie en Flux
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Stem cells
DNA/ Cell cycle - Sorting
600
Side Population (bone marrow of mouse)
Hoechst 33342
(laser UV or violet) emission : blue AND red
Apo
G0/G1
S
G2+M
G0/G1
events
Muscle
Hematopoietic
Apo.
S
G2+M
0
Adipocytes
0
Osteoblastes
33%
47%
14%
6%
Propidium Iodide
Overlay after
sorting
1024
G0/G1
events
Endotheliales
S
G2+M
Detection based on efflux Hoechst 33342
Propidium Iodide
Service Imagerie Cellulaire et Cytométrie en Flux
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Service Imagerie Cellulaire et Cytométrie en Flux
M.C. Gendron
A. Munier-01/2013
Multicolor analysis
Multicolor
6 coulors
26 population
CD3 FITC
CD38 PE
CD45 PerCP-C5.5
CD4 PE-Cy7
CD19 APC
CD8 APC-Cy7
after sorting
Service Imagerie Cellulaire et Cytométrie en Flux
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A. Munier-01/2013
Multiplex
A. Munier-01/2013
Detection of small events
 Ex : Cytometry Bead Area – BD Biosciences
 assays on beads
 New cytometers
Different Intensity of
fluorescence read on FL3
aIL-8-PE
FL3 - Beads
FL3 - Beads
aIL-1b-PE
aIL-6-PE
FL2 - PE
 Detection of small particles
(0.2 µm) according to their size and their fluorescences and of
intensity of their fluorescence
aIL-10-PE
aTNF-a-PE
aIL-12-PE
Ann Biol Clin, vol. 67, n°4, juillet-aout 2009
sensitivity, optic, electronic, informatic
 Fluorescences probes
FL2 - PE
Service Imagerie Cellulaire et Cytométrie en Flux
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Service Imagerie Cellulaire et Cytométrie en Flux
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Detection of microorganisms
Detection of microorganisms
Autofluorescence
Resolution of multiple small species by forward and side scatter
Chlorophylle
Phycoerythrine
Phycocyanine
Picoeucaryotes
Synechococcus
Prochlorococcus
Service Imagerie Cellulaire et Cytométrie en Flux
Service Imagerie Cellulaire et Cytométrie en Flux
A. Munier-01/2013
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Marine Biology
Medical Biology
Crucial role of microorganisms in the functioning of earth’s biosphere
No Antibiotic
Antibiotic added
FS vs SS
FS vs SS
Examples of phytoplancton population analysis coming from
different coastal ecosystems with each cell being identified by
their light scattering properties (size) and by their red
chlorophyllfluorescence excited with a 488 nm laser.
Light scatter cytograms of E.coli cells before and after addition of ampicillin.
Data obtained from, Bio-Rad, USA.
Service Imagerie Cellulaire et Cytométrie en Flux
Service Imagerie Cellulaire et Cytométrie en Flux
A. Munier-01/2013
A. Munier-01/2013
Food Biology
Plant Biology
DNA in plants
Detection of Listeria in milk products with CMF
using fluorescent antibodies
automatical analysis methods in milk products
Listeria monocytogenes
Not just pathogenics ...
Viability studies of beer yeast used in australian
brewing industry
Service Imagerie Cellulaire et Cytométrie en Flux
A. Munier-01/2013
S.J. Ochatt
Service Imagerie Cellulaire et Cytométrie en Flux
A. Munier-01/2013
Supplementary methods
A hybrid
Flow cytometry
Microscopy
Quantification
+++
+/-
Discrimination
individual
individual
Visualization
-
+++
Photobleaching
-
+++
Reproductibility
+++
+
Observed events
∞
Some hundreds
Cell sorting
+++
+/-
Complementarity
YES
YES
 The ImageStream is a high-speed automated microscope that
captures images in flow (low rate)
detector
Spectral
decomposition
element
cells in flow
Brightfield
illuminator
autofocus
laser
ImageStreamX
Amnis
Service Imagerie Cellulaire et Cytométrie en Flux
Service Imagerie Cellulaire et Cytométrie en Flux
A. Munier-01/2013
Technological innovation
Velocity
detector
A. Munier-01/2013
Technological innovation
 LEAP is an imaging cell sorter (shooting laser)
 Attune a cytometer an acoustical focusing
Attune
Life Technologies
Schematic of acoustic focusing principle.
LEAP
Cyntellect
Service Imagerie Cellulaire et Cytométrie en Flux
Service Imagerie Cellulaire et Cytométrie en Flux
A. Munier-01/2013
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Technological innovation
Perspective
 CyTOF :
metal-labeled cells are introduced individually into an Inductively Coupled Plasma
Cells are atomized and ionized in an argon plasma
Atomic ions are extracted into the ion optics and Time-Of-Flight region
They are separated by mass and counted
Quantum Dots, are semiconductors whose electronic characteristics are
closely related to the size of individual crystal.
Excitation par UV
Analysis of up to 100 stable isotope labels in a single cell
No overlap between detection channels, no need to compensate
No autofluorescence
An unlabeled cell is invisible to the system
1000 cells/ sec
Efficiency of the cell intruction system 20-30%
Data output in text and FCS standards
CyTOF® system
DVS Sciences
Bandura, D.R., et al.
Anal Chem 81 (16): 6813-6822, 2009.
The smaller dot, the closer it
is to the blue of the
spectrum, and the larger dot,
the closer to the red. Dots
can exciting by U.V or
another laser.
Quantum Dot Corporation
Service Imagerie Cellulaire et Cytométrie en Flux
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Service Imagerie Cellulaire et Cytométrie en Flux
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Recent technological developments
Conclusion
 Sensitivity increase and multiparameters
 « ELISA on beads » for detection and quantification of several
products in the same sample
 CMF is a technology turned to the polychromy
Panel of cytokines in plasma
Receptor in supernatant or in cell lysate
RNA messengers « microarrays » on beads
 Sensitivity increase and number of analyzed parameters open
prospects into studies of microparticules
Current technological developments
 QuantumDots (nanocristaux)
Service Imagerie Cellulaire et Cytométrie en Flux
Service Imagerie Cellulaire et Cytométrie en Flux
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Documentation
Documentation
 Books and journals
 Articles
La cytométrie en flux. X. Ronot, D. Grunwald, J.F. Mayol & J. Boutennat.
Tec et Doc. Lavoisier. Paris. 2006.
Hoffman RA,Wang L, Bigos M, NolanJP : NIST/ISAC standardization study :
variability in assignment of intensity values to fluorescence standard beads
and in cross calibration of standard beads to hard dyed beads. Cytometry . 81A :
785-796. (2012)
Practical flow cytometry. Shapiro H.M.
Zbigniew Darzynkiewicz : Critical Aspects in Analysis of Cellular DNA
Content. Curr Protoc Cytom. 2011 April; CHAPTER: Unit–7.2. (2011)
4ème édition. 2003. Wiley-Liss ed.
Roderer : Optimizing a multicolor immunophenotyping assay.
485. (2007)
Cycle cellulaire et cytométrie en flux. Grunwald, J.F. Mayol & X. Ronot.
Tec et Doc. Lavoisier. Paris. 2010.
Cytometry : http://eu.wiley.com/WileyCDA/WileyTitle/productCd-CYTO.html
Service Imagerie Cellulaire et Cytométrie en Flux
Clin Lab Med 27 : 469-
Roederer : Quantum dot semiconductor nanocrystal for immunophenotyping
by polychromatic flow cytometry. Nat Med. Aug ; 12(8) : 972-7. (2006)
Maecker HT, Trotter J : Flow Cytometry Controls, Instrument Setup, and
the Determination of Positivity.. Cytometry 69A : 1037-42. (2006)
Perfetto, S. P., Chattopadhyay, P. K. and Roederer, M. : "17-Color Flow
Cytometry: Unraveling the Immune System." Nat Rev Immunol 4: 648-655. (2004)
Robert P. Wersto, et al : Doublet Discrimination in DNA Cell-Cycle Analysis.
Cytometry. 46A : 296-306. (2001)
Service Imagerie Cellulaire et Cytométrie en Flux
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SPACE
Documentation
 Web cytometry
Association Française de Cytométrie : http://afcytometrie.fr
OCEAN
International Society for Analytical Cytology : http://isac-net.org/
Life technologies : http://www.invitrogen.com/site/us/en/home.html
 Analyse cellulaire Flow cytometry  resources  flow cytometry tutorials
EARTH
 List of discussion
TARA
Liste de discussion : http://www.cyto.purdue.edu/
 Free software
WEASEL : http://www.wehi.edu.au/cytometry/WEASEL.html-net.org/
Service Imagerie Cellulaire et Cytométrie en Flux
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Service Imagerie Cellulaire et Cytométrie en Flux
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