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......the same applies to Cell Biology
Cell Surface Marker:
Fluorochrome labeledantibodies
enable the characterizationof thecellular PHENOTYPE either byFlow
Cytometry or Imaging (e.g. CD4:CD8 Ration, HIV Patients)
Membrane:
Membrane analysis ist oftenused in the fieldof:
Ion Chanel and Transport
Structural integrity
Apoptosis and Necrosis
Live/Dead
Flow Cytometry or Imaging application
Nucleus:
DNA-specific and -reactive Dyes can be used
for DNA content analysis for:
Cell Cycle Analysis
Live/Dead Detection
Proliferation
and can be detected either
by Flow Cytometry or Imaging
IL-12
INF-y
IL-4
Intra Cellular Analysis:
Fluorochrome labeledantibodies enable the
characterization of Intracellular Structresand/or Proteins e.g. Cytokines and can be
detected either by Flow Cytometry or
Imaging (e.g. IL-12, INF-y uponst activation)
Cellular Analysis Flow Cytometry Applications
DNA (cell cycle analysis, proliferation)
Intracellular staining (cytokines, antigens)
Functionality tests
Proliferation
Apoptosis
Redox Potential
pH
Phagocytosis
Ion Indicators (e.g. Calcium)
Surface staining (Phenotyping)
Cell Membrane (Structural Integrity)
Flow Cytometry , Clinical Aspekts
Stem Cells (CD34)
DNA content (ploidity)
Multi Drug Resistance (MDR)
Cytokine Detection (IC, EC)
Subtype Classification (e.g. Treg, DC)
Crossmatching
Phenotyping (e.g.Transplantation [HLA], HIV)
CancerFlow Cytometry
� Cell Analysis
� Subclass Isolation
� Single Cell Suspension
� Fluorescence Based
� Fluid Stream
� Single Cell Measurement
� Size of the Cell
� Granularity of the Cell
� Fluorescence
2/26/2013
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Sheath Fluid
Light SourceDetection
Laminar
Flow
Laminar
Flow
Sheath Fluid
SAMPLE
PMTPhoto
Multiplier
Tube
A/DDatatranslation
Data
Analysis
The Flow Cytometry Principle
Analyzed Parameters:
Forward Scatter (FSC)
Size of the cell
Side Scatter (SSC)
Granularity of the cells
Fluorescence
Intensity
Number of Events
Fl-1, Fl-2, Fl-3, Fl-4 etc.
LASER LIGHT (e.g. 488nm)
The Flow Cytometry Principle
The The Scatter Scatter Principle (FSC, SSC)Principle (FSC, SSC)
Right Angle Light Detector
Side Scatter (SSC)
Cell surface, Granularity
Forward Light Detector
Forward Scatter (FSC)
Cell size
Light Source
Forward Scatter (FSC)
Sid
e S
catt
er
(SS
C)
Granulocytes
Monocytes
Lymphocytes
The Forward/Side Scatter Principle (FSC, SSC)
Dot Blot
Sample; Lysed Whole Blood
granularitycell surface
Relative Size
What is a Dot BlotWhat is a Dot Blot• Depicts individual events (particles or cells) versus two detected parameters
• A standard Dot Blot used in Flow Cytometry is the so called Scatter Blot,consisting of the Forward Scatter (FSC) Information and the Side Scatter(SSC)
• Therefore each “Dot” on a Scatter Blot stands for a measured event(typically Cells) and its SSC value and FSC value.
• Another typically used parameter in a Dot Blot is Fluorescence Intensity
• In a standard assay a typical number of events measured is 100.000
Flu
ore
sce
nce
Inte
nsi
ty
Fluorescence IntensityForward Scatter (FSC)
Sid
e S
catt
er
(SSC
)
What is a Quadrant BlotWhat is a Quadrant Blot
• A Quadrant Blot (QB) is a Dot Blot, segmented into four sections
• The QB is an ideal tool to analyze ( as %) individual events versus twoparameters
• Typically this tool is used to analyze populations versus two fluorochromes(targets)
LL
UR
LR
UL
UL = upper left
UR = upper right
LL = lower left
LR = lower right
2/26/2013
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Data Acquisition, HistogramData Acquisition, Histogram
Time
Vo
lta
ge
T ime
Vo
lta
ge
T ime
Vo
lta
ge
Sample FlowVoltage Pulse in PMT
Peak:
Height
Width
Area
Laminar
Flow
Sheath Fluid
SAMPLE
What is a Histogram PlotWhat is a Histogram Plot
• The Histogram is a graphical representation showing a visual impression of the distribution of data, here Fluorescence Intensity
• The Fluorescence Intensity is proportional to the number of Binding sites
• The Peak symmetry (height/width=area) can also be used to analyse
• The reference here is the unstained cell (ideally using an Isotype Control)
15
The Flow Cytometry Workflow
Sample Collection
Sample Analysis
Cell Viability
Sample Preparation Extracellular Staining
Intra Cellular Staining
Block Unspecific Binding
Cytokines/Growthfacors/Hormons
DNA fragmentation
Cell Functionality
FOR INTERNAL USE ONLY
Block Unspecific Binding
Fc-Blocking Reagents
• Block non-specific Fc-mediated binding of antibodies
• Reduce background and improve resolution of flow cytometry data
Inhibit the non-specific Fc-gamma receptor (FcγR)
� optimal staining of selective antibody
� optimal signal to noise ration (decrease background)
eBioscience Blocking Reagents:
Human FcγR-Binding Inhibitor
anti Mouse CD16/32-Block Fc-Binding
Intra-/Extra-cellular Staining
Lysis of Red Blood Cells
� Reduced Backround
� Optimized Signal to Noise
� Optimized Scatter Plot
Intra-/Extra-cellular Staining
Intracellular Staining Buffers
� Maintain Scatter Characteristics after Fix/Perm
� No effect on targeting structure
� Cytosolic Proteins
� Cytokines
� Nuclear Factors
� Transcription Factors
� Optimized access to target structure (Antibody access)
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� Simultaneous analysis of surface molecules and intracellular antigens
� Single cell level
� 1st Surface Stain
� 2nd Fixation (to stabilise structure)
Formaldehyd mediated
� 3rd Permeabilisation (to allow access for mAb)
Saponin mediated
� Stimulation required due to low initial levels of target protein by resting cells
� PMA (phorbol ester, protein kinase activator)
� Ionomycin (calcium ionophore)
� anti-CD3
� Lipopolysaccharide (LPS)
� T-cell stimulation to produce INF-γ, TNF-α, IL-2 and IL-4
� PMA, Ionomycin
� anti CD3
� Monocyte stimulation to produce IL-6, IL-10 or TNF-α
� LPS
� Transport Inhibitors (block secretion)
� Monensin
� Brefeldin A
The Flow Cytometry Workflow
Sample Collection
Sample Analysis
Cell Viability
Sample Preparation Extracellular Staining
Intra Cellular Staining
Block Unspecific Binding
Cytokines/Growthfacors/Hormons
DNA fragmentation
Cell Functionality
The eBioscience Knowledge Center
www.eBioscience.com
The eBioscience Knowledge Center
www.eBioscience.com
The eBioscience Knowledge Center
www.eBioscience.com
The eBioscience Knowledge Center
www.eBioscience.com
2/26/2013
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The eBioscience Knowledge Center
www.eBioscience.com
Detection of Detection of Antigens (EC/IC)Antigens (EC/IC)
Specific Probe or Antibody
Dye/Fluorochrome
Introduction to Fluorescence TechniquesIntroduction to Fluorescence Techniques
Fluorescence DetectionFluorescence Detection
Four essential elements of fluorescence detection systems can be
identified from the preceding discussion:
1) Excitation source
2) Fluorophore
3) Wavelength filters
to isolate emission photons from excitation photons
4) Detector
that registers emission photons and produces a recordable output,
usually as an electrical signal or a photographic image. Regardless of
the application, compatibility of these four elements is essential for
optimizing fluorescence detection.
Fluorescence InstrumentationFluorescence Instrumentation
Fluorescence instruments are primarily of four types, each providing distinctly different information:
Spectrofluorometers and microplate readers
measure the average properties of bulk (µL to mL) samples.
Fluorescence microscopes
resolve fluorescence as a function of spatial coordinates in two or three dimensions for microscopic objects (less than ~0.1 mm
diameter).
Fluorescence scanners
including microarray readers, resolve fluorescence as a function of spatial coordinates in two dimensions for macroscopic objects
such as electrophoresis gels, blots and chromatograms.
Flow cytometers
measure fluorescence per cell in a flowing stream, allowing
subpopulations within a large sample to be identified and quantitated.
......it’s all about fluorescence.............it’s all about fluorescence.............it’s all about fluorescence.............it’s all about fluorescence.............it’s all about fluorescence.............it’s all about fluorescence.............it’s all about fluorescence.............it’s all about fluorescence.......
The Fluorescence Process
• Fluorescence is the result of a three-stage process
• Fluorescence occurs in rigid molecule structures
• Fuorescent molecules are in general: polyaromatic hydrocarbons or heterocycles
• These molecules are called fluorophores or fluorescent dyes.
• The fluorescence process is illustrated by the simple electronic-state diagram
_(Jablonski diagram)
Phenolphthalein
Flexible molecule, non-fluorescent
Fluorescein
Rigid molecule, fluorescent
2/26/2013
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The Fluorescence Process: Jablonski Diagramm
Stage 1 : Excitation (external source, idealy at the Exitation maximum)
Stage 2 : Excited-State Lifetime (finite 1-10ns)
Stage 2 : collision quenching
fluorescence resonance energy transfer (FRET)
intersystem crossing
Stage 3 : Fluorescence Emission
Tandem ConjugatesTandem Conjugates
Fluorescence Resonance Energy Transfer (FRET)
R-PE, PerCp, APC Cy 5, Cy 5.5, Cy 7, Alexa Fluor 700
488nm
Excitation
Far Red
EmissionDonor
Dy e
Acceptor
Dy e
Energy Transfer
Fluorescent Dyes in Immune Diagnostic
Multicolour Analysis = Multiple Parameters
34
Fluorescence IntensityFluorescence Intensity
Nu
mb
er
of
eve
nts
Fluorescent intensity
100 102101 104103 105
Fluorescence Intensity is proportional to the number of binding sites
35
Fluorescence Intensity and BrightnessFluorescence Intensity and Brightness
Relative Brightness:
PE - APC – PerCP- PE-Cy5.5 - PE-Cy5 - PE-TR - PE-Cy7 - APC-Cy7 - FITC
Bright Dim
Nu
mb
er
of
eve
nts
Fluorescent intensity
100 102101 104103 105
Defining bright versus dim: Stain IndexDefining bright versus dim: Stain Index
Stain Index (SI) = D/W
D = difference between pos and neg peak medians
W = 2 x robust SD
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Fluorescence Intensity and BrightnessFluorescence Intensity and Brightness
1 – 2 – 3 – 4 – 5 low- - - - - - - - - - - - - - - - -high
Fluorescent DyesFluorescent Dyes
FFluoresceinluoresceinIIsosoTThiohioCCyanatyanat (FITC)(FITC)
Xanthendye
excitation: 488 nmemission max.: 518 nm
molecular weight: 0.389 kDa
Detected in FL-1 channel in most instruments
Prone to Photobleaching
Intensity sufficient for
strong expressed AG
Staining of human platelets with purified
mouse IgG1 isotype control (cat.#14-4714)
(dotted histogram) or purified HIP8 (solid
histogram) followed by FITC anti-mouse IgG
(cat.#11-4011 ). Total viable cells were used for
analysis.
1 – 2 – 3 – 4 – 5 low- - - - - - - - - - - - - - - - -high
AAlexalexa FFluorluor®® 488488 (AF 488)(AF 488)
Modified Xanthendye
excitation: 488 nmemission max.: 518 nm
molecular weight: 0.643 kDa
Detected in FL-1 channel in most instruments
Excellent Photostability
Staining of HeLa cells with Alexa
Fluor® 488 Mouse IgG2a, K Iso Cntrl
(cat. 53-4724) (open histogram) or
Alexa Fluor® 488 anti-human
MICA/M ICB (6D4) (colored
histogram). Total viable cells were
used for analysis.
OH2
N
SO3
-SO
3
-
NH2
+
CO2
-
O
O
N
O
O
1 – 2 – 3 – 4 – 5 low- - - - - - - - - - - - - - - - -high
RedRed--PPhycohycoEErythrinrythrin (R(R--PE)PE)
Phycobiliprotein(Cyanobacteria)
Oscillatoria rubescens
excitation: 488 nm
emission max.: 575 nm
molecular weight: > 240 kDa
Very bright signal, high quantum yield
Prone to FotobleachingStaining of permeabil ized C57Bl/6
thymocytes (left) and permeabil ized Jurkat
(right) cells with 0.5 µg of PE Mouse IgG1,
K Iso Cntrl (cat. 12-4719) (open histogram)
or 0.5 µg of PE 1E7.2 (colored histogram).
Total cells were used for analysis. 1 – 2 – 3 – 4 – 5 low- - - - - - - - - - - - - - - - -high
2/26/2013
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PE PE PE PE –––– Texas Red (PETexas Red (PETexas Red (PETexas Red (PE----TR)TR)TR)TR)
excitation: 488 nm
emission max.: 615
molecular weight: > 240 kDa +TR
Detected in FL3 on single laser
instruments
PE overlap requires compensation
PE PE PE PE –––– AlexaAlexaAlexaAlexa Fluor 610 (PEFluor 610 (PEFluor 610 (PEFluor 610 (PE----AF610)AF610)AF610)AF610)
excitation: 488 nm
emission max.: 628
molecular weight: > 240 kDa +AF610
Alternative to PE-TR
Less overlap into PE channel due to
628nm emission
Less subject to Fc mediated binding
than PE-Cy5
1 – 2 – 3 – 4 – 5 low- - - - - - - - - - - - - - - - -high
1 – 2 – 3 – 4 – 5 low- - - - - - - - - - - - - - - - -high
PEPE--Cy5Cy5 (Cy(Cy--ChromeChrome®®, TRI, TRI--COLORCOLOR®®, PC5, PC5®®))
Tandem Conjugate: R-PE and Cy5
excitation: 488 nm
emission max.: 670 nm
molecular weight: R-PE + X Cy5 > 242 kDa
Detected in FL3 or FL4 in most instruments
When used with APC in dual laser instruments,
inter laser compensation is required
Staining of normal human peripheral blood
cells with PE-Cy5 Mouse IgG1, K Iso Cntrl
(cat. 15-4714) (open histogram) or PE-Cy5
anti-human CD2 (RPA-2.10) (colored
histogram). Total cells were used for
analysis.
1 – 2 – 3 – 4 – 5 low- - - - - - - - - - - - - - - - -high
PE PE PE PE ---- Cy5.5Cy5.5Cy5.5Cy5.5
excitation: 488 nm
emission max.: 690 nm
molecular weight: R-PE + X Cy5 > 242 kDa
Detected in FL3 or FL4 in most
instruments
When used with APC in dual laser
instruments, inter laser compensation is required
Better choice for those instruments than
PE-Cy5
Pe rCPPe rCPPe rCPPe rCP ---- Cy5.5Cy5.5Cy5.5Cy5.5
excitation: 488 nm
emission max.: 690 nm
molecular weight: R-PE + X Cy5 > 242 kDa
Detected in FL3 or FL4 in most
instruments
When used with APC in dual laser
instruments, inter laser compensation is required
Better choice for those instruments than
PE-Tandems due to the larger stokes shift
1 – 2 – 3 – 4 – 5 low- - - - - - - - - - - - - - - - -high
1 – 2 – 3 – 4 – 5 low- - - - - - - - - - - - - - - - -high
PerCPPerCP--eFluor™710eFluor™710
Anti-mouse CD4 (clone RM4-5), anti-mouse CD8 (clone 53-6.7), and anti-human
CD3 (clone OKT3) were conjugated to either PerCP-eFluor™ 710 (red) or PerCP-
Cy5.5 (pink) for direct comparison. Mouse splenocytes were stained with anti-
CD4 (left panel) or anti-CD8 (middle panel). Human PBMCs were stained with
anti-CD3 (right panel).
Comparison of PerCP-eFluor™ 710 and PerCP-Cy5.5 Conjugates
Detected in FL3 on most instruments; tandem dye,
resonance energy transfer from PerCP molecule to
eFluor® 710; when used with APC on dual laser
machines, needs a cytometer capable of inter-laser
compensation. Better choice for dual laser
instruments than PE-tandems as a result of its large
stokes shift. 2-3 fold brighter than PerCP-Cy5.5.
1 – 2 – 3 – 4 – 5 low- - - - - - - - - - - - - - - - -high
PEPE--Cy7 Cy7 (Phycoerythrin(Phycoerythrin--Cyan7 Tandem Conjugate) Cyan7 Tandem Conjugate)
Tandem Conjugate: R-PE and Cy7
excitation: 488 488 488 488 nm nm nm nm or 530 nm530 nm530 nm530 nm
emission max.: 773 nm
molecular weight: R-PE + X Cy5 > 242 kDa
Detected in FL3 or FL4 in most instruments
Issues:
Brightness, Compensation, Stability-Fixation
eBioscience Improved PE-Cy7 Conjugations:
Brighter Staining
Less Compensation
Greater Stability
1 – 2 – 3 – 4 – 5 low- - - - - - - - - - - - - - - - -high
The histogram demonstrates staining with new
PE-Cy7 anti-human CD14 from eBioscience (red
line), or from the competitor (green line). Data
provided courtesy of Mihoko Whalen, Tobias
Kollmann and Pascal Lavoie from the Child &
Family Research Institute in Vancouver.
AAllolloPPhycohycoCCyaninyanin (APC)(APC)
Phycobiliproteinfound in blugreen algae
excitation: 633 / 635 nmemission max.: 660 nm
molecular weight: 105 kDa
Intensity sufficient for low expressed antigens
Staining of C57Bl/6 bone marrow cells with
0.125 μg of APC Rat IgG2b Isotype Control (cat.
17-4031) (blue histogram) or 0.125 μg of APC
anti-mouse Gr-1 (RB6-8C5 ) (purple histogram).
Total viable cells were used for analysis.
1 – 2 – 3 – 4 – 5 low- - - - - - - - - - - - - - - - -high
2/26/2013
9
AAlexalexa FFluorluor®® 647 (AF 647)647 (AF 647)
Carboxylic Acid, Succinimidyl Ester
excitation: 633 / 635 nmemission max.: 665 nm
molecular weight: 1.250 kDa
AF 647 Intensity comparable to APC
Intracellular staining
Photo stability
Staining of F9 embryonal
carcinoma cells with 0.5 μg of
Alexa Fluor® 647 Rat IgG2a Iso
Cntrl (cat. 51-4321) (open
histogram) or 0.5 μg of Alexa
Fluor® 647 anti-mouse OCT3/4
(EM92) (colored histogram). Total
cells were used for analysis. 1 – 2 – 3 – 4 – 5 low- - - - - - - - - - - - - - - - -high
eFluoreFluor®® 660 (alternative to APC or 660 (alternative to APC or AlexaAlexa FluorFluor®® 647) 647)
Human per iphera l blood monocytes w ere
stained with mous e Ig M isotype control
(dotted line) or the anti-CD36 clone NL07
conjugat ed to eFluor 660 (red histogram) or
Alexa Fluor 647 (gray histogram).
1 – 2 – 3 – 4 – 5 low- - - - - - - - - - - - - - - - -high
Immunofluorescent i ma ge of cryos ection
of mouse intestine stain ed with eFluor
660 conjugated anti-mouse L YVE-1
antibody and nuclear counterstain DAPI.
excitation: 633 / 635 nmemission max.: 660nm
Excellent Photo stability
Comparable brightness to APC
Ideal tool for IHC applications
APC-eFluor® 780(bright and stabile alternative for APC(bright and stabile alternative for APC--Cy7 or APCCy7 or APC--H7)H7)
Human PBMC
Anti CD3 (UCHT1)
APC-eFluor 780
Human PBMC
Anti CD3 (UCHT1)
APC-Alexa Fluor 750
Overlay Panel 1/2 Mouse splenocytes
Anti CD8 (53-6.7)
APC-eFluor 780 (blue)APC-H7 (red)
1 – 2 – 3 – 4 – 5 low- - - - - - - - - - - - - - - - -high
Fluorochromes for BD FACSCanto IITM
(4-2-2 Configuration)
Excitation [nm]: 488nm488-nm solid state, 20-mW
Filter: 530/30 BP
Filter: 585/42 BP
Filter: 670 LP
Filter: 780/60 BP
FITC (Fluorescein)
Alexa FluorTM 488
PE (Phycoerythrin)
Alexa FluorTM 555
PerCP-eFluor 710
PerCP
PE-Cy 7
Excitation [nm]: 633nm633-nm HeNe, 17-mW laser
Filter: 660/20 BP
Filter: 780/60 BP
APC
Alexa FluorTM 647
eFluorTM 650NC
APC-eFluor 780
APC-Cy7
Fluorochromes for BD FACSCanto IITM
(4-2-2 Configuration)
Excitation [nm]: 405nm405-nm solid state diode, 30-mW
Filter: 450/50 BP
Filter: 502-525nm
eFluorTM 450
Pacific BlueTM
Fixable Viability
Dye eFluor 505
Fluorochromes for BD FACSCanto IITM
(4-2-2 Configuration)
2/26/2013
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Blue Laser OptionsBlue Laser Options RedRed Laser OptionsLaser Options
2010 | www.eBioscience.com57
Green Laser OptionsGreen Laser Options Fluorochromes example for a 7 color stain using the red
and blue laser line
How can we go further???
What about a 10 colour stain????
EMPOWER YOUR VIOLET LASER!!!
The Next Generation in FluorescenceThe Next Generation in Fluorescence
eFluoreFluor™ ™ NanocrystalsNanocrystals
2/26/2013
11
eFluoreFluorTMTM Nanocrystal CompositionNanocrystal Composition
� Core composition and size determines color of the Nanocrystal
� Inorganic shell improves stability and brightness
� Unique, propriartity lipid coating provides water solubility and functional
groups for conjugation
� Biomolecules are covalently attached to functional groups on coated
nanocrystal
Core Shell Lipid Layer
2- 10 nm
CdSe ZnS Water Soluble Nanocrystal
Semiconductor Semiconductor NanocrystalsNanocrystals; The principal ; The principal
The excited complex is a very strong reductant� photobleaching
The shell keeps the excited electron from interacting with the environment
A high-energy photon excites an
electron across the bandgap
A bandgap-energy photon is emitted as
the electron falls back to the ground stage
Semiconducting
Infinite Solid
Conduction Band
(LUMO)
Valence Band
(HOMO)
Bandgap (Eg)
Antibonding orbital
(mostly metal d character)
Bonding orbital
(mostly anion p character)
Eg(0), α and β are material constants
Semiconducting
Nanocrystal
eFluoreFluor NanocrystalNanocrystal Platform TechnologiesPlatform Technologies
Flow
Fl. MicroscopyIn Vivo
eFluoreFluor™ ™ NanocrystalsNanocrystals
• Large “Stokes Shift”
• Narrow emission = minimal compensation
• Excellent photostability
• Compatible with conventional fluorochromes/ Filter sets
Excitation/EmissionEmission Comparison
Tra
dit
ion
al
Tra
dit
ion
al
eF
luo
rS
olu
tio
n
The eFluor™ Solution: Compensation Requirements
VioletViolet Laser OptionsLaser Options
2/26/2013
12
Fluorochromes example for a 10 color stain
using the violet, red and blue laser line Conclusions:Conclusions:
• Plan your experiment, what do I want to know
• Know your cells
• Consider environmental conditions
• Sensitivity required
• Selection of dyes
• Select instrument; acquisition rate/flow rate
• Optimize staining procedure
• Titrate you antibody for optimal staining
• Data analysis
Multicolor Multicolor Flow Flow Cytometry:Cytometry:
Experimental DesignExperimental Design
Design and analysis of multicolor flow Design and analysis of multicolor flow
cytometry experimentscytometry experiments
• Design
• Instrument configuration
• Fluorochrome performance
• FluorPlan™ Spectra Viewer
• Tandem dye considerations
• Compensation considerations
• Analysis
• Eliminating false positives
• Proper controls for gating
Instrument considerationsInstrument considerations
• Instrument configuration
• Lasers, PMTs, filters and dichroic mirrors
�This will define your fluorochrome choices
• Obtain baseline PMT voltage settings
�Use only as a place to start
• Use fluorescent calibration beads to understand
resolution and performance for each detector
available
Assessment of detectors on an LSR II Assessment of detectors on an LSR II
using 8using 8--peak beadspeak beadsFITC: PE: PerCP-Cy5.5: PE-Cy7:
Blue laser (488 nm)
APC: APC-eFluor 780:Red laser (633 nm)
eFluor 450: eFluor 605NC:Violet laser (405 nm)
2/26/2013
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Fluorochrome performanceFluorochrome performance
• Understand fluorochrome performance
• Match brightest fluorochromes with:
�antigens expressed at low density
�antigens with non-uniform expression
�antigens with unknown expression patterns
• Adjust instrument settings for optimal resolution
of targets
Defining bright versus dim: Stain IndexDefining bright versus dim: Stain Index
Stain Index (SI) = D/W
D = difference between pos and neg peak medians
W = 2 * robust SD
Relative brightness of fluorochromes Relative brightness of fluorochromes
using CD4using CD48-peak beads in:
Combining detector & fluorochrome Combining detector & fluorochrome
performance: assessment of dim antigensperformance: assessment of dim antigens
FITC detector PE-Cy7 detector APC-eFluor® 780
detector
eFluor® 450
detector
CD4 FITC CD4 PE-Cy7 CD4 APC-eFluor® 780 CD4 eFluor® 450
PBMC stained for CD4:
Optimizing instrument settings:Optimizing instrument settings:
PerCPPerCP--Cy5.5 & PECy5.5 & PE--Cy7 exampleCy7 example
0
200
400
600
800
1000
1200
350 450 550 650 750 850
Flu
ore
sce
nce
In
ten
sity
(arb
itra
ry u
nit
s)
Emission Wavelength (nm)
PerCP-Cy5.5 PE-Cy7
Optimizing instrument settings for B220 Optimizing instrument settings for B220
PerCPPerCP--Cy5.5 & CD3 PECy5.5 & CD3 PE--Cy7Cy7
CD
3 P
E-C
y7
B220 PerCP-Cy5.5
CD3 PE-Cy7
B220 PerCP-Cy5.5
CD
3 P
E-C
y7
B220 PerCP-Cy5.5
B220 PerCP-Cy5.5 single stain
PerCP-Cy5.5 detector
626 volts
PerCP-Cy5.5
spillover in the
PE-Cy7 detector
669 volts
626 volts 520 volts
CD3 PE-Cy7
669 volts
520 volts
80%
12%
2/26/2013
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Design and analysis of multicolor flow Design and analysis of multicolor flow
cytometry experimentscytometry experiments
• Design
• Instrument configuration
• Fluorochrome performance
• FluorPlan™ Spectra Viewer
• Tandem dye considerations
• Nanocrystal considerations
• Compensation considerations
• Analysis
• Eliminating false positives
• Proper controls for gating
Use Use FluorPlanFluorPlan™ to determine ™ to determine
fluorochromefluorochrome optionsoptions
2011 | www.eBioscience.com80
FluorPlanFluorPlan™ Spectra Viewer™ Spectra Viewer
2011 | www.eBioscience.com
FluorPlanFluorPlan™ Spectra Viewer™ Spectra Viewer
Design and analysis of multicolor flow Design and analysis of multicolor flow
cytometry experimentscytometry experiments
• Design
• Instrument configuration
• Fluorochrome performance
• FluorPlan™ Spectra Viewer
• Tandem dye considerations
• Compensation considerations
• Analysis
• Eliminating false positives
• Proper controls for gating
Using tandem dyes in multicolor flow Using tandem dyes in multicolor flow
cytometry experimentscytometry experiments
• Tandem Dye Consideratons
• Loss of FRET efficiency
�Fixation – use freshly prepared, high quality
formaldehyde
�Light exposure
• Shelf life
2/26/2013
15
Fixation & Fixation & photostabilityphotostability of PEof PE--Cy7Cy7
Freshly stained
& analyzed cells
Fixed cells:
2% Formaldehyde
30 minutes
Room temp
Fixed cells:
2% Formaldehyde
overnight
4⁰C
Live cells:
Ambient light
6 hours
Room temp
PE detector
Fixation & Fixation & photostabilityphotostability of APCof APC--eFluor® 780eFluor® 780
Fixed cells:
2% Formaldehyde
30 minutes
Room temp
Fixed cells:
2% Formaldehyde
overnight
4⁰C
Live cells:
Ambient light
6 hours
Room temp
APC detector
Freshly stained
& analyzed cells
Additional tandem dye considerationsAdditional tandem dye considerations
• Vendor specific manufacturing protocols
• Compensation requirements can vary
between conjugations (different antibodies)
and even different lots of the same antibody
• Compensation for tandem dye conjugates
should ALWAYS be set with the antibody used
in the staining panel
Design and analysis of multicolor flow Design and analysis of multicolor flow
cytometry experimentscytometry experiments
• Design
• Instrument configuration
• Fluorochrome performance
• FluorPlan™ Spectra Viewer
• Tandem dye considerations
• Nanocrystal considerations
• Compensation considerations
• Analysis
• Eliminating false positives
• Proper controls for gating
Designing a multicolor flow cytometry Designing a multicolor flow cytometry
experimentexperiment
• Minimize spillover
• Consider compensation issues when assigning
fluorochromes to antigens
�Avoid spillover from a bright population into a
detector requiring high sensitivity
• Optimize the core markers of your staining panel for
best resolution
Design and analysis of multicolor flow Design and analysis of multicolor flow
cytometry experimentscytometry experiments
• Design
• Instrument configuration
• Fluorochrome performance
• FluorPlan™ Spectra Viewer
• Tandem dye considerations
• Nanocrystal considerations
• Compensation considerations
• Analysis
• Eliminating false positives
• Proper controls for gating
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Analyzing a multicolor flow cytometry Analyzing a multicolor flow cytometry
experimentexperiment
• Singlet gating and excluding dead cells
• Critical for the elimination of false positives
• Appropriate controls for gating
• FMO (Fluorescence Minus One)
• Isotype controls
• Internal negative
Principle of singlet gating
2011 | www.eBioscience.com92
Sig
na
l in
ten
sity
time
Single-cell
laser
Sig
na
l in
ten
sity
time
Doublet
laser
Practice of singlet gatingPractice of singlet gatingCD3+ CD19+ double-positive cells?
Value of singlet gatingValue of singlet gating
No! Just 2 cells stuck together.
Dead cells interfere with optimal Dead cells interfere with optimal
staining resolutionstaining resolution
2011 | www.eBioscience.com
Fresh thymocytes
Anti-CD3-stimulated thymocytes
96 2011 | www.eBioscience.com
SingletsViable
Eliminating false positives with singlet Eliminating false positives with singlet
gating & exclusion of dead cellsgating & exclusion of dead cells
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Analyzing a multicolor flow cytometry Analyzing a multicolor flow cytometry
experimentexperiment
• Singlet gating and excluding dead cells
• Critical for the elimination of false positives
• Appropriate controls for gating
• FMO (Fluorescence Minus One)
• Isotype controls
• Internal negative
Negative spreading phenomenonNegative spreading phenomenon
Fluorescence Minus One (FMO)Fluorescence Minus One (FMO)
• In multicolor experiments, compensation can
introduce error, resulting in “negative
spreading”
• The FMO control is a sample containing every
fluorochrome-conjugated Ab but one
• Including appropiate reference e.g. Isotype
control
2010 | www.eBioscience.com100
CO
MP
EN
SA
TIO
N
CO
MP
EN
SA
TIO
N
Isotype controls: Ubiquitous…Isotype controls: Ubiquitous…
• An Ab of the same isotype conjugated to the
same fluorochrome
Rat IgG2a PE
Human PBMC were stimulated for 5 hrs
with PMA & ionomycin in the presence
of brefeldin. CD3+CD4+ cells were used
for analysis
Autofluorescence (lymphocytes)
Rat IgG2a PE (isotype)
Unstimulated cells
Stimulated cells
IL-2 PE
Limitations of the isotype controlLimitations of the isotype control
• It is its own protein
– Although it is the same isotype, it is still a completely different Ab molecule
• Separate conjugation reaction
– F/P can vary slightly from reaction to reaction
• Isotypes were originally generated & evaluated for non-binding to surface proteins
– How they behave intracellularly can be unpredictable
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Multicolor flow cytometry: SummaryMulticolor flow cytometry: Summary
• Know your instrument capabilities & adjust settings to optimize performance
– Minimize compensation
– Maximize stain index
– Be aware of negative spreading
• Choose markers & fluorochromes to maximize stain index for each marker
– May take a couple tries for more complex panels
• Eliminate false positives (singlets & dead cells)
• Include relevant controls (isotype, FMO, etc.)
Apoptosis, Necrosis, Viability, Vitality, Functionality.......
Apoptosis from A to Z
105eBioscience Confidential | eBioscience.com
Apoptosis
• Greek: apo – from, ptosis – falling
• One of the main types of Programmed Cell Death (PCD)
• Multi-biochemical event (incl. cell to cell interaction)
• Leading to cell death; save dispose of the cell
• In contrast; Necrosis is uncontrolled cell death from acute cellular injury
• On going increase in Apoptosis research since the early ’90
• Many focus areas such as uncontrolled proliferation => Cancer
• As an example, Apoptosis is needed in the differentiation of fingers during ..development of the embryo
~50 to 70 billion of cells undergo apoptosis every day
(average human adult)
In one year the mass of the cells undergoing apoptosis is
~equal to the weight of the individual body
106eBioscience Confidential | eBioscience.com
Apoptosis vs Necrosis
107eBioscience Confidential | eBioscience.com
Induction and Regulation of Apoptosis
Induction
Cell repair (e.g. DNA damage due ionizing radiation )
Infection (e.g. v irus)
Cell stress (e.g. starvation)
Homeody namics (homeostasis)
Control:
Cell signals
Hormones
Grow th factors
Cy tokines
Nitric ox yde
Regualtion
cell-cell interaction (e.g. haematopoesis)
108eBioscience Confidential | eBioscience.com
Apoptosis Inducers in Cell Biology
Actinomycin DCamphtothecin
CycloheximideDexamethasone
Etoposide
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109eBioscience Confidential | eBioscience.com 110eBioscience Confidential | eBioscience.com
Normal Cell
Apoptotic Body (AB) Formation
Lysis of AB
Continued Blebbing
Cell shrinkage Chromatin Condensation
Membrane Blebbing
Nuclear Collapse
The Apoptosis Process
111eBioscience Confidential | eBioscience.com
Apoptosis induced Cell function changes
Mitochondrial
Transition Pore opening
Phosphatidyl
Serine (PS) Translocation
Caspase Activity
Metabolic Activity
DNA
Condensation
Plasmamembrane
Integrity
DNA
Fragmentation
Normal Cell
Lysis of AB
Mitochondrial
Membrane Potential
Normal Cell
Lysis of AB
112eBioscience Confidential | eBioscience.com
Mitochondrial membrane potential
• JC-1 is a membrane permeable dye for flow cytometry and fluorescent microscopy.
• Selectively enters the mitochondria where it reversibly changes color as membrane potentials .. ..
..increase (over values of 80-100 mV).This property is due to the reversible formation of J-aggregates upon membrane polarization.
• Excitation: 488 nm @ 59%
498 nm @ 82%
592 nm @ 100%
• Emission (max): 530 nm (JC-1 monomer)
590 nm (J-aggregate)
• JC-1 is both qualitative, in regards to the shift from green to orange fluorescence emission, ..and.quantitative, as measured by fluorescence intensity, in both filter sets.
JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'- tetraethylbenzimidazolylcarbocyanine iodide)
113eBioscience Confidential | eBioscience.com
Apoptosis induced Cell function changes
Mitochondrial
Transition Pore opening
Phosphatidyl
Serine (PS) Translocation
Caspase Activity
Metabolic Activity
DNA
Condensation
Plasmamembrane
Integrity
DNA
Fragmentation
Normal Cell
Lysis of AB
Mitochondrial
Membrane Potential
Normal Cell
Lysis of AB
Cytochrome C Release
114eBioscience Confidential | eBioscience.com
Cytochrome C detection
Cytochrome C Release
MitochondrialTransition Pore
opening
MitochondrialMembrane Potential
Human Cytochrome c Platinum ELISA
Cytochrome C detection via MonoclonalAntibodies
� Flow Cytometry
� Western Blot
� Immuno Histochemistry
� Imaging
� Immuno Precipitation
� Functional Assays
� ELISA
� Human� Mouse� Rat
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Fura-2 AM• Preferred dye for ratiometric imaging microscopy with digital image analysis• Upon binding Ca2+, the excitation spectrum of Fura-2 shifts to shorter
..wavelengths (400 to 300 nm)
• Peak emission remains steady (~510 nm)• Peak excitation: depending on free Ca2+conc. between 300nm and 400nm
• Peak emission: 510 nm• Molecular weight: 1001.86 Da
Indo-1 AM• single excitation / dual emission dye• Unbound Indo-1 has a peak emission at 485 nm, which shifts to 410 nm
upon Ca2+ binding. In Flow Cytometry, this shift can be measured over time
and represented as a ratio of the two emission wavelengths.• Peak Excitation: 346 nm
• Peak Emission: 475 nm (Unbound Indo-1 =485nm shift to 410nm dep. on Ca2+ binding)
• Molecular Weight: 1009.91
Calcium Sensor Dye eFluor® 514• Indicator for intracellular free calcium
• Detection by: FC, IHC, Imaging, Microplate Readers• Increased cellular uptake � increased brightness
• Excitation: 488 nm
• Emission: 514 nm• Calcium binding affinity: Kd = 232 nM
• MW: ~1100 Da
• Not recommended for quantitative measurements
Calcium Sensor Dye eFluor® 514
Jurkat cells were harvested, w ashed and loaded with
Calcium Sensor D ye eFluor® 514 for 30 minutes at 37°C.The left panel shows c ells that were w ashed and analyzed
by flow cytom etry unstimulated (b lue histogram) or
stimulated with 1 ug/mL ionomycin (purp lehistogram).The
right panel shows Jurkat cells loaded with Calcium Sensor
Dye eFluor® 514 that were acquired on a flow cytom eterfor 1 minute and then removed for theaddit ion of 1 ug/mL
ionomycin and immediately p laced back on the flow
cytometer for continued acquisit ion..
Calcium Sensing Dyes
116eBioscience Confidential | eBioscience.com
Apoptosis induced Cell function changes
Mitochondrial
Transition Pore opening
Phosphatidyl
Serine (PS) Translocation
Caspase Activity
Metabolic Activity
DNA
Condensation
Plasmamembrane
Integrity
DNA
Fragmentation
Normal Cell
Lysis of AB
Mitochondrial
Membrane Potential
Normal Cell
Lysis of AB
Phosphatidyl Serine (PS) Translocation
117eBioscience Confidential | eBioscience.com
Untreated Treated
10µM Camptothecin, 4h
Detection of Phosphatidyl serine (PS) translocationvia Annexin V labeling
Cell M
em
bra
ne
CytosolPhosphatidyl serine
Control:
Counterstain with cell
impermeant DNA-dyee.g. PI, 7AAD
Normal C ell Early Apoptosis Late Apoptosis Necrosis
Formats@eBioscience:
eFluor 450
FITCR-PE
PerCp-eFluor 710
APC
PE-Cy7
Biotin
118eBioscience Confidential | eBioscience.com
Annexin V FITC
&
Propidium iodid
Etopodise treated
Thymocytes
MAG 40x MAG 100xMAG 40x
Detection of Phosphatidyl serine (PS) translocationvia Annexin V labeling
119eBioscience Confidential | eBioscience.com
Apoptosis induced Cell function changes
Mitochondrial
Transition Pore opening
Phosphatidyl
Serine (PS) Translocation
Caspase Activity
Metabolic Activity
DNA
Condensation
Plasmamembrane
Integrity
DNA
Fragmentation
Normal Cell
Lysis of AB
Mitochondrial
Membrane Potential
Normal Cell
Lysis of AB
CaspaseActivity
120eBioscience Confidential | eBioscience.com
Caspase activity
C aspase 1 (pc) H WB, IP, IHC
C aspase 2L p12 H,M,R WB, IP, IHC
C aspase 3 (pro) M WB
C aspase 3 (pc) H WB, IP, IHC
C aspase 7 H WB
C aspase 8 H WB
C aspase 10 H WB
C aspase 11 M FC
C aspase 12 H,M,R WB
C aspase 12 M WB, IP, IHC, FC
C aspase 13 H,M,R WB
WB: Western Blotting
IP: Immuno Precipitation
IHC: Immuno His to Chemis try
FC: Flow Cytometry
H: Human
M: Mouse
R: Rat
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121eBioscience Confidential | eBioscience.com
CaspGLOW™
• Detection of active Caspases in live cells• Caspase inhibitors are conjugated to FMK
� provide cell-permeability� non-toxic
� irreversible binding• Detection in Flow Cytometry and Imaging• One hour procedure
Data provided by:
Simple Protocol
Collect cells and resuspend in
incubation buffer
Add caspase probe and Incubate
@37C for 20min
Analyze
122eBioscience Confidential | eBioscience.com
Apoptosis induced Cell function changes
Mitochondrial
Transition Pore opening
Phosphatidyl
Serine (PS) Translocation
Caspase Activity
Metabolic Activity
DNA
Condensation
Plasmamembrane
Integrity
DNA
Fragmentation
Normal Cell
Lysis of AB
Mitochondrial
Membrane Potential
Normal Cell
Lysis of AB
MetabolicActivity
123eBioscience Confidential | eBioscience.com
Metabolic activity (Viability dyes)
• Calcein is a green fluorescent viability dye excited @ 488nm • Acetoxymethyl ester (AM) makes the molecule hydrophobic and suitable for ..uptake across cell membranes in live cells• Calcein AM becomes highly fluorescent when the acetoxymethyl ester is..hydrolyzed by cytoplasmic esterases thereby releasing the hydrophilic highly ..fluorescent cell-bound calcein• Since dead cells do not contain active esterases, dead cells are not labeled• Calcein AM is not toxic so it can also be used for short-term cell tracing studies
• Calcein Blue AM is a UV excited alternative to Calcein AM, having an excitation ..similar to DAPI, Hoechst, and AMCA
• Calcein Violet 450 AM, is a violet laser (405 nm) excited equivalent to Calcein ..AM. Co-staining with Annexin V or 7-AAD is recommended to allow the greatest ..resolution between live and dead/ apoptotic cells
• Propidium iodide (PI) and 7-amino-actinomycin D (7-AAD) are both ..fluorescent viability dyes that can be used to measure plasma membrane ..integrity; only cells with compromised plasma membranes will stain with PI and ..7-AAD
124eBioscience Confidential | eBioscience.com
Metabolic activity (Cell Tracking and Proliferation)
Cell Tracking & Proliferation Dye
• CFSE [5-(and 6)-C arboxyfluorescein diacetate succinimidyl ester]
• C ell tracking and proliferation studies
• Also used in CTL assays and cell motility studies
• C FSE readily crosses intact cell membranes, inside the cells, intracellular esterases cleave the
..acetate groups to yield the fluorescent carboxyfluorescein molecule. The succinimidyl ester
..group reacts with primary amines, crosslinking the dye to intracellular proteins.
• Cell division can be measured as successive halving of the f luorescence intensity of .. ..
. C FSE
125eBioscience Confidential | eBioscience.com
Metabolic activity (Cell Tracking and Proliferation)
Cell Tracking & Proliferation Dye
• CPD eFluor™670: Cell Proliferation Dye eFluor™ 670• Cell tracking and proliferation studies• Also used in CTL assays and cell motility studies• Excitation 633nm; Emission 670nm (APC Filter 660/20 BP)• Works similar than CFSE• Compatible with GFP and YFP detection• For in vivo and in vitro applications
126eBioscience Confidential | eBioscience.com
Metabolic activity (Cell Tracking and Proliferation)
Cell Tracking & Proliferation Dye
• CPD eFluor™450: Cell Proliferation Dye eFluor™ 450• Cell tracking and proliferation studies• Also used in CTL assays and cell motility studies• Excitation 405nm; Emission 450nm (450/50 BP)• Works similar than CFSE• Compatible with most reporter proteins• For in vivo and in vitro applications
• Mouse splenocytes • 10 uM CPD eFluor® 450
• cultured for 3 days
• with ConA (blue histogram) • without ConA (purple histogram)
• Splenocytes (Thy1.1 mice) • 10 uM CPD eFluor® 450
• Injected into C57Bl/6 mice (purple histogram)
• Injected into B6D2F1 mice (blue histogram)• Splenocytes collected 72 hours after injection
• Unlabeled cells (Gray)
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Cell Proliferation detection via BrdU
BrdU(Bromodeoxyuridine)
• Detection of proliferation (S-phase)
• BrdU is a synthetic nucleoside analog of Thymidin• Thymidin will be replaced by BrdU during replication
Simplif ied
Protocoll
Safes up to an 1h
time compared to
conventional
methods
128eBioscience Confidential | eBioscience.com
Simultaneous Measurement combining Proliferation Dyes:
CFSE or Cell Proliferation Dyes
Immunophenotyping; surface markers
Annexin V
Live/Dead discrimination;7AAD, FVD
Nuclear stains; BrdU, transkription factors
Cytokine expression
The staining is always depended on the cell type. Fixation and Permeabilisation can effect performance. Some buffer might effect performance.
129eBioscience Confidential | eBioscience.com
Apoptosis induced Cell function changes
Mitochondrial
Transition Pore opening
Phosphatidyl
Serine (PS) Translocation
Caspase Activity
Metabolic Activity
DNA
Condensation
Plasmamembrane
Integrity
DNA
Fragmentation
Normal Cell
Lysis of AB
Mitochondrial
Membrane Potential
Normal Cell
Lysis of AB
P lasmamembrane Integrity
130eBioscience Confidential | eBioscience.com
Plasma membrane integrity
life/dead discrimination using cell impermeant dyes
Mouse t hymocytes were prep ared as a single ce ll
suspensio n a nd incubated over night at 37 °C inmedium (left ) or medium wit h 1µM de xamet hasone(right). Cel ls wer e harvested and sta ine d us ing the
Annexin V FIT C Apopt osis Detect ion Kit and Prop id iumIodide Staining Solution (cat. 00-6990).
Propidium Iodide (PI)
• Exclusion of nonviable cells in flow cytometric analysis
• Binds to double stranded DNA by intercalating between ..basepairs
• Is excluded from cells with intact plasma membranes
• Can be used in FL3 for inviability exclusion, but should be ..analyzed in FL2 when used as a counterstain for FITC
..Annexin V.
7-amino-actinomycin D (7-AAD)
• Exclusion of nonviable cells in flow cytometric analysis.
• Can be used in place of PI (propidium iodide) to reduce ..wavelength spill over in Multicolour experiments
• Can be used in combination with PE (phycoerythrin) and
..FITC (fluorescein isothiocyanate) conjugated antibodies • Fluorescence is detected in the far red range of the
..spectrum (650 nm long-pass filter).
7AAD stain o n human L ymphocytes after f reeze/t haw
cycle
7AAD
15%
131eBioscience Confidential | eBioscience.com
Life/Dead discrimination; Fixed Cells
Fixable Viability Dyes are a viability dyes that can be used to irreversibly label dead cells prior to:
� Cryopreservation
� Fixation
� Permeabilization
Unlike 7AAD and propidium iodide, cells labeled with Fixable Viability Dyes can be washed, fixed,
permabilized, and stained for intracellular antigens without any loss of staining intensity of the dead
cells. Thus, using Fixable Viability Dyes allows dead cells to be excluded from analysis when intracellular targets are being studied. Fixable Viability Dyes may be used to label cells from all species.
Fixable Viability Dye eFluor® 450
Fixable Viability Dye eFluor® 506
Fixable Viability Dye eFluor® 660
Fixable Viability Dye eFluor® 780
132eBioscience Confidential | eBioscience.com
Amine-reactive fluorescent reagents Binds free amine groups on proteins
Unlabeled cells
Labeled cells� Intact membrane
Labeled cells� Compromised membrane
NO
fluorescence
MEDIUM
fluorescence
BRIGHT
fluorescence
Life/ Dead discrimination; Fixed Cells
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133eBioscience Confidential | eBioscience.com
Amine-reactive fluorescent reagents Binds free amine groups on proteins
NO
fluorescence
MEDIUM
fluorescence
BRIGHT
fluorescence
Life/ Dead discrimination; Fixed Cells
Fixable Viability Dye eFluor® 450
Fixable Viability Dye eFluor® 506
Fixable Viability Dye eFluor® 660
Fixable Viability Dye eFluor® 780
134eBioscience Confidential | eBioscience.com
Nuclear Labeling of DNA
�anthraquinone dye
�high affinity for double-stranded DNA�membrane-permeable dye
�nuclear DNA content analysis eg. for ploidy /cell cycle analysis because it binds DNA stoichiometrically� In fluorescent microscopy, it can be used as a nuclear counterstain.
For cell cycle/DNA analysis applications in order to optimize to optimize the CV for the G1 and G2/M peaks a higher wavelength filter is recommended:
710LP, 735LP dichroic mirror and 780/60 band pass),
Do not combine with far-red fluorochromes excited by the 488 or 633 nm laser lines
(PE-Cy7, PerCP-eFluor® 710, PerCP-Cy5.5, APC, Alexa Fluor® 647, Alexa Fluor® 700, APC-eFluor® 780)
Cat. 65-0880
Nuclear Red (DRAQ 5™)
Ex: 488-647nm
Ex: 488nm
Detect: P erCP-eFluor® 710 or P erCP-Cy5.5
(685LP dichroic mirror and 710/50 BP)
Ex: 633nm
Detect: APC (660/20 band pass)C57Bl/6 bone marrow cells were stained with FITC anti-mouse CD45 (30-F11) (cat.
11-0451) (left) and eFluor® 450 anti-mouse TER-119 (cat. 48-5921) (right), followed by staining with 5 µM Nuclear RED (DRAQ5™) for 15 minutes at room temperature. Total viable cells were used for analysis.
135eBioscience Confidential | eBioscience.com
Nuclear Labeling of DNA
� anthraquinone dye
�high affinity for double-stranded DNA
�membrane-permeable dye
� In flow cytometry, it can be used to distinguish nucleated and non-nucleated cells
� In fluorescent microscopy, it can be used to identify and discriminate the nucleus and cytoplasm
…without the need for a second dye due to its high intensity staining of the nucleus and low intensity
…staining of the cytoplasm.
Please make sure that your instrument is capable of detecting this dye.
Cat. 65-0881Nuclear ORANGE (CyTRAK Orange™) Ex: 488-550nm Ex: 488nm Em max: 610nm (610/20 BP)
136eBioscience Confidential | eBioscience.com
Cell Tracking; CellVue® dye
�lipophilic dyes
�labeling of the cell membrane
�identifying and tracking of labeled cells
�rapid and stable labeling
�Multiplexable
�Supplied with diluent and labeling vehicle
C ellVue® Lavender (Cat.88-0873) Ex: 420 Emmax: 461
C ellVue® Jade (Cat.88-0876) Ex: 478 Emmax: 508
C ellVue® NIR780 (Cat.88-0875) Ex: 633 Emmax: 776
C ellVue® Maroon (Cat.88-0870) Ex: 647 Emmax: 667
C ellVue® Plum (Cat.88-0871) Ex: 652 Emmax: 671
C ellVue® Burgundy (Cat.88-0872) Ex: 683 Emmax: 707
C ellVue® NIR815 (Cat.88-0874) Ex: 786 Emmax: 814
137eBioscience Confidential | eBioscience.com
Apoptosis induced Cell function changes
Mitochondrial
Transition Pore opening
Phosphatidyl
Serine (PS) Translocation
Caspase Activity
Metabolic Activity
DNA
Condensation
Plasmamembrane
Integrity
DNA
Fragmentation
Normal Cell
Lysis of AB
Mitochondrial
Membrane Potential
Normal Cell
Lysis of AB
DNA Fragmentation
138eBioscience Confidential | eBioscience.com
• Detection of apoptotic cells by flow cytometry• 2-color staining method• Labeling DNA breaks and total cellular DNA
The kit content:
� Instructions, all reagents required, positive and negative control� Washing, reaction and rinsing buffers� Terminal deoxynucleotidyl transferase enzyme (TdT)� Fluorescein-deoxyuridine triphosphate (dUTP)� Propidium iodide / RNase A solution for counterstaining the total DNA
One of t he most easil y measured features of apoptotic cells is t he break-up of t he genomic DNA by cell ular
nucl eases. These DNA fragments c an be extract ed from apoptotic cells and res ult in t he appearance of DNAladderi ng w hen t he DNA is anal yzed by agarose gel electrophoresis. The DNA of non-apopt otic c ells t hat
remains largel y i ntact does not displ ay t his l adderi ng on agaros e gels duri ng el ectrophoresis. The large
number of DNA fragments appearing in apoptotic cells results in a multitude of 3' -hydroxyl termini i n t he
DNA. This propert y can be used to identi fy apoptotic cells by l abeling t he 3'-hydroxyl ends with directly
conjugated fluorescein- deoxyuri di ne tri phosphat e nucleotides (FITC-dUTP). The enzyme terminaldeoxynucl eotidyl trans ferase (TdT) catal yzes a templat e-i ndependent addition of deoxyribonucleoside
triphosphat es to the 3'-hydroxyl ends of double- or si ngle-stranded DNA wit h eit her blunt, recessed or
overhanging ends. A substantial number of t hese sites are available i n apoptotic cells providing the basis for
the method utiliz ed in the APO-DIRECT™ Kit. Non-apoptotic c ells do not inc orporat e signi ficant amounts of
the FITC-dUTP due to the lack of exposed 3'-hydroxyl DNA ends.
DNA break vs total DNA: APO-DIRECT™
APO-DIRECT is a trademark of Phoenix Flow Systems, San Diego, California.
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139eBioscience Confidential | eBioscience.com
• Anti-ssDNA stain of condensed chromatin• Staining of cells and/or tissue sections with• APOSTAIN followed by heat treatment, induces DNA denaturation in situ only in..apoptotic nuclei.• In the presence of formamide, only apoptotic nuclei DNA becomes denatured and..detectable• Applicable for Human, Mouse and Rat samples• For Flow Cytometry• For IHC staining including formalin fixe, paraffin embedded tissue sections
APOSTAIN™
DAPI counterstain APOSTAIN positive nuclei
140eBioscience Confidential | eBioscience.com
Apoptosis induced Cell function changes
Mitochondrial
Transition Pore opening
Phosphatidyl
Serine (PS) Translocation
Caspase Activity
Metabolic Activity
DNA
Condensation
Plasmamembrane
Integrity
DNA
Fragmentation
Normal Cell
Lysis of AB
Mitochondrial
Membrane Potential
Normal Cell
Lysis of AB
141eBioscience Confidential | eBioscience.com
The eBioscience Knowledge Center
www.eBioscience.com
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The eBioscience Knowledge Center
www.eBioscience.com
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The eBioscience Knowledge Center
www.eBioscience.com
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The eBioscience Knowledge Center
www.eBioscience.com
2/26/2013
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145eBioscience Confidential | eBioscience.com
…..perfectionperfectionperfectionperfection isisisis finallyfinallyfinallyfinally attainedattainedattainedattained
notnotnotnot whenwhenwhenwhen theretheretherethere isisisis nononono longerlongerlongerlonger anythinganythinganythinganything totototo add,add,add,add,
butbutbutbut whenwhenwhenwhen theretheretherethere isisisis nononono longerlongerlongerlonger anythinganythinganythinganything totototo taketaketaketake
awayawayawayaway ............
Antoine de Saint Exupéry (1900-1944), Terre des Hommes (1939)
146eBioscience Confidential | eBioscience.com
Thank You