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Cytometry: Cell analysers and cell sorting
Staff : H. Fohrer-Ting et E. Devevre.
Characteristics
Caracteristics
LSR II
Cell sorting
Blue Laser 488nm
x
YG Laser 561nm
Table of content:
Page 1: Characteristics
Page 2: Applications
Page 3: Cell sorting and
statistics
Page 4: Good practices
Aria III
BD
Influx
x
x
x
x
x
x
Red Laser 633nm
x
x
x
Violet Lasers 405nm
x
x
x
Nb of parameters
11
15
17
DIVA
DIVA
BD
Sortware
Sort device: tubes
4 ways
6 ways
Sort device: plate
x
x
Sort device: slide
x
x
Software
Index sort
x
About the technique:
• 
• 
• 
• 
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Rare event detection from live cell
suspension
High sensitivity
Phenotypic analysis as well as functional
studies
Purification of cell population for
downstream applications
Kinetic analysis (calcium flux..)
Associated software:
Both software allow doublet exclusion and
automated compensations.
Staff assistance for
technology
development, panel
design, data
analysis
Standardisation:
• 
• 
• 
Export template experiment with analysis/
sorting gating strategy.
Daily quality control.
Application setup standardization ensures
consistency of results overtime across
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multiple systems.
Cytometry: Cell analysers and cell sorting
Staff : H. Fohrer-Ting et E. Devevre.
Applications
•  Multicolor analysis
Table of content:
Page 1: Characteristics
Page 2: Applications
Page 3: Cell sorting and
statistics
•  Cell cycle
Page 4: Good practices
<Blue 675-715-A>: KI67
10 5
28.3
10 4
3.03
2.43
Combined use of DAPI (xaxis) and Ki67 (y-axis)
allows identification of the
different cell cycle stages.
10 3
62.4
10 2
0
0
50K
100K
150K
200K
<Violet 425-475-A>: DAPI
250K
•  Microparticles
Global(BD
Sheet1 Influx)
Megamix 0,5 µm
Megamix 0,9 µm
Megamix 3,0 µm
MP 0,1 à 1,0 µm
Global Sheet1
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MP
FITC-A
Printed on: Fri Mar 27, 2015 02:50:29 CET
Global Sheet1
Printed on: Fri Mar 27, 2015 02:50:29 CET
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Printe
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Cytometry: Cell analysers and cell sorting
Responsable : H. Fohrer-Ting et E. Devevre.
Cell sorting
•  Cell sorting
Flexible cell sorting conditions for improved cell
recovery
Purify cell populations up to 99,5%.
•  Index sort (BD Influx)
Table of content:
Page 1: Characteristics
Page 2: Applications
Index sort allows the reviewing of the
phenotype of every cell sorted into a multiposition device, such as a 96-well plate.
The created fcs file contains all the sort
deposition and tray position information on an
event-by-event basis.
Page 3: Cell sorting and
statistics
Page 4: Good practices
•  Downstream applications
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• 
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Cell culture
Protein extraction
RNA extraction
Gene and protein array analysis
•  Statistics
Sort and analysis statistics can be exported and
further analyzed on third-party software.
Cytometry: Cell analysers and cell sorting
Staff : H. Fohrer-Ting et E. Devevre.
Good practices
•  Distinction of several cell
subpopulations on the combined 2 size
criteria: FSC and SSC.
10
5
SSC-A
10 4
Table of content:
5.35
10
3
10 2
0
0
Page 1: Characteristics
Page 3: Cell sorting and
statistics
Page 4: Good practices
100K
150K
FSC-A
200K
250K
Left: Linear scale. PBMC: distinction of lymphocytes,
monocytes and granulocytes population.
Right: Log scale, Tumor sample: distinction of the infiltrating
lymphocytes.
•  Doublet exclusion. Doublets are
caracterised by a higher width pulse, thus
an increased Area pulse.
250K
FSC-A versus FSC-H.
Cells outside the gates
have a higher Area
are excluded.
200K
150K
FSC-H
Page 2: Applications
50K
80.6
100K
50K
0
0
50K
100K
150K
FSC-A
200K
250K
Dead cell exclusion. Multiple detectors
allow a large choice of dead cell marker.
Markers
are
Sheet1such as DAPI, PI or 7AADPage
1 of DNA
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intercalant. Dead cells will stain positively for
these dies. They can be used only on non-fixed
cells.
Other dies calls live-dead stains are specific of
the amine group present at the membrane or in
the cell cytoplasm. Dead cells will display an
increase staining intensity.
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