2/26/2013 1 2 ......the same applies to Cell Biology Cell Surface Marker: Fluorochromelabeledantibodies enable thecharacterizationofthe cellular PHENOTYPE either by Flow Cytometry or Imaging (e.g. CD4:CD8 Ration, HIV Patients) Membrane: Membrane analysis ist often used in the field of: 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 contentanalysis 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 Structres and/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) Cancer Flow Cytometry Cell Analysis SubclassIsolation Single Cell Suspension Fluorescence Based Fluid Stream Single Cell Measurement Size of the Cell Granularity of the Cell Fluorescence
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2/26/2013
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2
......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
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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
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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
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2/26/2013
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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
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
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The Fluorescence Process: Jablonski Diagramm
Stage 1 : Excitation (external source, idealy at the Exitation maximum)
• 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.
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
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
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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
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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
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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
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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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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
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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
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Metabolic activity (Cell Tracking and Proliferation)
• 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
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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
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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
The staining is always depended on the cell type. Fixation and Permeabilisation can effect performance. Some buffer might effect performance.
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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
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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%
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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
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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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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
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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
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.
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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.
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
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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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• 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