Applications Flow Cytometry
• Genetically encoded fluorescent reporter proteins
(transfection-, infection-, induction- rates, expression, deletion…)
• Immunostainings of cell-specific antigens
(extra- and intracellular)
• DNA stainings
(cell cycle, ploidy, apoptosis/necrosis)
• Functional assays and Kinetics
(apoptosis, cell proliferation, cell signaling: Ca2+-flux)
used by BioOptics with permission of Tanja Winkler
GFP
Genetically encoded fluorescent proteins report for …
•
•
•
Transfection
Infection …
GFP
Expression
Induction
Deletion …
23%
Protein interaction (FRET, split-GFP)
Gene
GFP
+Dox
Location (Photoactivatable GFP)
dsRed
•
GFP
used by BioOptics with permission of Tanja Winkler
dsRed
Genetically encoded fluorescent proteins report for …
•
•
Split-GFP
Transfection
Infection …
B
Protein A
Expression
Induction
Deletion …
CGFP
NGFP
•
Protein interaction (FRET, split-GFP)
•
Location (Photoactivatable GFP)
GFP
used by BioOptics with permission of Tanja Winkler
Genetically encoded fluorescent proteins report for …
•
Transfection
Infection …
•
Expression
Induction
Deletion …
•
Protein interaction (FRET, split-GFP)
•
Location (Photoactivatable GFP)
405nm (1P)
830nm (2P)
+
CO2
488nm
Victora et al 2011
used by BioOptics with permission of Tanja Winkler
SSC-A
Immunophenotyping by cell-specific surface staining
Live cells
SSC-H
FSC-A
Singlets
Empty ch
SSC-W
-Auto
-fluorescence
Fluorescent ch
Bone Marrow
used by BioOptics with permission of Tanja Winkler
CD11c-BV421
Ter119-BV510
Gr1-APC
C-Kit-PerCP-Cy5.5
Ly6c-Pe
Live, Singlets
CD11b-PeCy7
Immunophenotyping by cell-specific surface staining
B220-Fitc
B220-Fitc
used by BioOptics with permission of Tanja Winkler
Ly6c-PE
Immunophenotyping: Multi-color FACS
X-color
Laser 1: 405nm
450/50
PacBlue
BV421
525/50
BV510
Laser2: 488nm
585/40
BV605
780/60
BV785
530/30
Fitc
AF488
GFP
695/40
PerCPCy5.5
Laser3: 561nm
582/15
PE
610/20
PECF594
Laser4: 640nm
780/60
PE-Cy7
670/30
APC
A647
780/60
APC-Cy7
4
x
x
x
x
5
x
x
x
x
x
6
x
x
x
x
x
x
7
x
x
x
x
x
x
x
8
x
x
x
x
x
x
x
x
9
x
x
x
x
x
x
x
x
x
10
x
x
x
x
x
x
x
x
x
x
11
x
x
x
x
x
x
x
x
x
x
x
Laser & filter settings: Fortessa
used by BioOptics with permission of Tanja Winkler
Y
Phenotyping and functionality by cytoplasmic-, nuclear
staining
•Staining of surface antigen
Intracellular stainings
(transcription factor)
•Staining of cytoplasmic
& nuclear antigens
Nuclear
Y
Y
•Permeabilization
Aiolos
Y
•Fixation
Igκ (cytoplasmic)
Cytoplasmic
B220-BV421 (surface)
used by BioOptics with permission of Tanja Winkler
http://www.cytobank.org/facselect/
DNA-content
Ploidy
dsDNA dye
PI
Propidium iodide
Excitation
Laser [nm]
Emission
[nm]
Notes
488/561
620
dsDNA/RNA
Fix/Perm
2n
488/561
645
dsDNA
Fix/Perm
DAPI
350/375
460
dsDNA
Fix/Perm
Hoechst 33342
350/375
460
dsDNA
Cellmembrane
permeable
3n
S
Sub-G1
G1/G0
M
7-AAD
7-Aminoactinomycin D
1n
Cell-cycle
DNA-content (linear)
used by BioOptics with permission of Tanja Winkler
G2
4n
G1/G0
G2
S-phase analysis by BrdU uptake
M
•In vitro/ in vivo Bromodeoxyuridine (BrdU) incorporation
42%
Time X
•Harvest cells/organ
•Staining of surface antigen
4%
BrdU-Fitc
•Fixation/Permeablization
•DNase-treatment
•Staining of BrdU and total DNA
7-AAD (DNA content)
Bone Marrow: 30 min
(alternative: Click-it EdU assay)
used by BioOptics with permission of Tanja Winkler
Live/dead discrimination with impermeable dyes
Live
DNA dye
Excitation [nm]
PI
488/561
7-AAD
488/561
DAPI
350
Protein dye
Excitation [nm]
LIVE/DEAD Fixable
(Molecular Probes)
350/405/488/561/640
Fixable viability dye eFluor
(eBioscience)
350/405/488/640
Zombie (BioLegend)
350/405/640
Propidium iodide
7-Aminoactinomycin D
Notes
Non-fixable
Notes
Fixable
…and many more
used by BioOptics with permission of Tanja Winkler
Dead
Apoptosis
Staining/dye
method
note
Annexin 5
Phosphatidylserine (PS) translocation to outer
membrane
No Fix/Perm
Ca2+ buffer
dsDNA dye:
PI, 7-AAD, DAPI
Membran impermeable
DNA-fragmentation (Sub-G1)
No Fix/Perm
Fix/Perm
Anti-pro-apoptotic
molecules
Active form of caspase-3
Cleaved PARP
Fix/Perm
JC-1
Mitochondrial Membrane Potential,
Emission:
530nm (monomer in cytoplasm, apoptotic)
590nm (aggregates in mitochondria, live)
No Fix/Perm
ratiometric
Apoptotic
Dead
Annexin V
Dead
PI
PI/7-AAD
Live
used by BioOptics with permission of Tanja Winkler
Live
Apoptotic
Annexin V-Fitc
Proliferation
Cell proliferation dyes
Excitation
[nm]
CFSE
488
nonfluorescent
ester
CellTrace Violet
(Molecular Probes)
405
amine-reactive
fluorescent molecule
(esterases activated)
eFluor 450/670
(eBioscience)
405/640
VPD450 (BD)
405
876543210
IgG1
FSC-A
…and many more
CellTrace Violet
used by BioOptics with permission of Tanja Winkler
Intracellular signaling: Ca2+-flux
Calcium
sensor dye
Excitation
Emission
eFluor 514
490nm
514nm
Indo-1
346nm
410 nm (Ca2+)
485nm
Flura-2
300nm (Ca2+)
400nm
510nm
Ca2+
Ca2+
Igβ
•Loading of cells with calcium sensor dye
•Acquisition & Stimulation at 37°C
Fortessa I (water bath )
eFluor 514
•Incubation at 37°C
Time
used by BioOptics with permission of Tanja Winkler
Ca2+
Ca2+
Ca2+ 2+
Ca
Ionomycin
Ca2+