...the same applies to Cell Biology 2 Cellular Analysis Nucleus: -specific and -reactivedyescan beused for contentanalysis for: Cell Cycle Analysis Live/Dead Detection Proliferation and can be detected either byflow Cytometry or Imaging Membrane: Membrane analysisist oftenusedin the fieldof: Ion Chanel and Transport Structural integrity Apoptosisand Necrosis Live/Dead FlowCytometry or Imagingapplication IL-12 INF-y IL-4 Intra CellularAnalysis: Fluorochromelabeledantibodiesenable the characterization ofintracellular Structres and/or Proteins e.g. Cytokinesand can be detected either byflow Cytometry or Imaging (e.g. IL-12, INF-y uponst activation) Cell SurfaceMarker: Fluorochrome labeledantibodies enable thecharacterizationofthe cellularphenotype either byflow Cytometry orimaging (e.g. CD4:CD8 Ration, HIV Patients) Flow Cytometry Applications Surface staining (Phenotyping) Intracellular staining (cytokines, antigens) (cell cycle analysis, proliferation) Cell Membrane (Structural Integrity) Functionality tests Proliferation Apoptosis Redox Potential ph Phagocytosis Ion Indicators (e.g. Calcium) Flow Cytometry, Clinical Aspekts Phenotyping (e.g.transplantation [HLA], HIV) Cancer Flow Cytometry Crossmatching content (ploidity) Stem Cells (CD34) Multi Drug Resistance (MDR) Subtype Classification (e.g. Treg, DC) Cytokine Detection (IC, EC) Cell Analysis Subclass Isolation Single Cell Suspension Fluorescence Based Fluid Stream Single Cell Measurement Size of the Cell Granularity of the Cell Fluorescence 1
The Flow Cytometry Principle SAMPLE Sheath Fluid Sheath Fluid The Flow Cytometry Principle Analyzed Parameters: Laminar Flow Laminar Flow Data Analysis A/D Datatranslation LASER LIGHT (e.g. 488nm) 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. Light Source Detection PMT Photo Multiplier Tube The Scatter Principle (FSC, SSC) The Forward/Side Scatter Principle (FSC, SSC) Dot Blot cell surface granularity Granulocytes Forward Light Detector Forward Scatter (FSC) Cell size Side Scatter (SSC) Monocytes Light Source Lymphocytes Right Angle Light Detector Side Scatter (SSC) Cell surface, Granularity Sample; Lysed Whole Blood Forward Scatter (FSC) Relative Size What is a Dot Blot Depicts individual events(particles or cells) versus two detectedparameters 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 eventsmeasured is 100.000 What is a Quadrant Blot A Quadrant Blot (QB) is a Dot Blot, segmentedinto four sections The QB is an ideal tool to analyze ( as %) individual events versus two parameters Typically this tool is used to analyze populations versus two fluorochromes (targets) UL UR Side Scatter (SSC) Fluorescence Intensity LL LR Forward Scatter (FSC) Fluorescence Intensity UL = upper left UR = upper right LL = lower left LR = lower right 2
Data Acquisition, Histogram Sheath Fluid SAMPLE Laminar Flow Sample Flow Voltage Pulse in PMT Voltage Time What 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 IsotypeControl) Voltage Time Voltage Time Peak: Height Width Area The Flow Cytometry Workflow Block Unspecific Binding Sample Preparation Sample Collection Block Unspecific Binding Extracellular Staining Intra Cellular Staining Cell Viability 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) Sample Analysis Cell Functionality optimal staining of selective antibody optimal signal to noise ration (decrease background) ebioscience Blocking Reagents: fragmentation Cytokines/Growthfacors/Hormons Human FcγR-Binding Inhibitor anti Mouse CD16/32-Block Fc-Binding 15 FOR INTERNAL USE ONLY Intra-/Extra-cellular Staining Lysis of Red Blood Cells 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) Reduced Backround Optimized Signal to Noise Optimized Scatter Plot 3
Simultaneous analysis of surface molecules and intracellular antigens Single cell level 1st SurfaceStain 2nd Fixation (to stabilise structure) Formaldehyd mediated 3rd Permeabilisation (toallow access for mab) Saponin mediated Stimulation required due to low initial levels of targetprotein by resting cells PMA (phorbol ester, protein kinaseactivator) Ionomycin (calcium ionophore) anti-cd3 Lipopolysaccharide (LPS) T-cellstimulation 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 Preparation Sample Collection Sample Analysis fragmentation Block Unspecific Binding Extracellular Staining Intra Cellular Staining Cell Viability Cell Functionality Cytokines/Growthfacors/Hormons The ebioscience Knowledge Center The ebioscience Knowledge Center www.ebioscience.com www.ebioscience.com The ebioscience Knowledge Center The ebioscience Knowledge Center www.ebioscience.com www.ebioscience.com 4
The ebioscience Knowledge Center Detection of Antigens (EC/IC) Specific Probe or Antibody Dye/Fluorochrome www.ebioscience.com Fluorescence Detection Introduction to Fluorescence Techniques 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 Instrumentation Fluorescence instruments are primarily of four types, each providing distinctly different information: Spectrofluorometers and microplate readers measure the averageproperties 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... The Fluorescence Process Fluorescence is the result of a three-stageprocess Fluorescence occurs in rigid molecule structures Fuorescent molecules arein general: polyaromatic hydrocarbons or heterocycles These molecules are calledfluorophores 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 5
The Fluorescence Process: Jablonski Diagramm Tandem Conjugates Fluorescence Resonance Energy Transfer (FRET) 488nm Excitation Donor Dy e Acceptor Dy e Far Red Emission Energy Transfer 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 R-PE, PerCp, APC Cy 5, Cy 5.5, Cy 7, Alexa Fluor 700 Fluorescence Intensity Fluorescent Dyes in Immune Diagnostic Fluorescence Intensity is proportional to the number of binding sites Number of events Multicolour Analysis = Multiple Parameters 10 0 10 1 10 2 10 3 10 4 10 5 Fluorescent intensity 34 Bright Fluorescence Intensity and Brightness Relative Brightness: PE -APC PerCP- PE-Cy5.5 -PE-Cy5 - PE-TR - PE-Cy7 - APC-Cy7 - FITC Dim Defining bright versus dim: Stain Index Stain Index (SI) = D/W Number of events 10 0 10 1 10 2 10 3 10 4 10 5 Fluorescent intensity 35 D = difference between pos and neg peak medians W = 2 x robust SD 6
Fluorescence Intensity and Brightness FluoresceinIsoThioCyanat (FITC) Fluorescent Dyes Xanthendye excitation: 488 nm emission max.: 518nm molecular weight: 0.389 kda Detected in FL-1 channel in most instruments Prone to Photobleaching 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 usedfor analysis. Intensity sufficient for strong expressed AG Alexa Fluor 488 (AF 488) H 2 N SO 3 - O SO 3 - NH 2 + Red-PhycoErythrin(R-PE) 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/MICB (6D4) (colored histogram). Total viable cells were used foranalysis. Modified Xanthendye excitation: 488 nm emission max.: 518 nm molecular weight: 0.643 kda Detected in FL-1 channel in most instruments Excellent Photostability O N O O O CO 2 - Staining of permeabilized C57Bl/6 thymocytes (left) and permeabilized 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). Phycobiliprotein(Cyanobacteria) Oscillatoria rubescens excitation: 488 nm emission max.: 575 nm molecular weight: > 240 kda Very bright signal, high quantum yield Prone to Fotobleaching Total cells were used for analysis. 7
PE-Cy5 Cy5(Cy-ChromeChrome, TRI-COLOR, PC5 ) PE Texas Red (PE-TR) excitation: 488 nm emission max.: 615 molecular weight: > 240 kda +TR Detected in FL3 on single laser instruments PE overlap requires compensation PE Alexa Fluor 610 (PE-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 Fcmediated binding than PE-Cy5 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. 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 PE - Cy5.5 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 PerCP- Cy5.5 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 PerCP-eFluor 710eFluor 710 Comparison of PerCP-eFluor 710 and PerCP-Cy5.5 Conjugates 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). 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. PE-Cy7 (Phycoerythrin-Cyan7 Tandem Conjugate) AlloPhycoCyanin yanin(apc) Tandem Conjugate: R-PE and Cy7 excitation: 488 nm or 530 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 Phycobiliproteinfound in blugreen algae excitation: 633 / 635 nm emission max.: 660 nm molecular weight: 105 kda Intensity sufficient for low expressed antigens The histogram demonstrates staining with new PE-Cy7 anti-human CD14 fromebioscience (red line),orfrom thecompetitor (green line). Data provided courtesy of Mihoko Whalen, Tobias Kollmann and Pascal Lavoie from the Child & FamilyResearchInstituteinVancouver. ebioscience Improved PE-Cy7 Conjugations: Brighter Staining Less Compensation Greater Stability 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. 8
Alexa Fluor 647 (AF 647) efluor 660 (alternative to APC or Alexa Fluor 647) AF 647 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 Carboxylic Acid, SuccinimidylEster excitation: 633 / 635 nm emission max.: 665 nm molecular weight: 1.250 kda Intensity comparable to APC Intracellular staining Photo stability cells were used for analysis. Human per ipheral 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). Immunofluorescent i mage 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 nm emission max.: 660nm Excellent Photo stability Comparable brightness to APC Ideal tool for IHC applications APC-eFluor 780 (bright and stabile alternative for APC-Cy7 Cy7 or APC-H7) Fluorochromes for BD FACSCanto II TM (4-2-2 Configuration) 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) Excitation [nm]: 488nm 488-nm solid state, 20-mW Filter: 530/30 BP Filter: 585/42 BP Filter: 670 LP Filter: 780/60 BP FITC (Fluorescein) Alexa Fluor TM 488 PE (Phycoerythrin) Alexa Fluor TM 555 PerCP-eFluor710 PerCP PE-Cy 7 Fluorochromes for BD FACSCanto II TM (4-2-2 Configuration) Fluorochromes for BD FACSCanto II TM (4-2-2 Configuration) Excitation [nm]: 633nm 633-nm HeNe, 17-mW laser Filter: 660/20 BP Filter: 780/60 BP APC Alexa Fluor TM 647 efluor TM 650 NC APC-eFluor 780 APC-Cy7 Excitation [nm]: 405nm 405-nm solid state diode, 30-mW Filter: 450/50 BP Filter: 502-525nm efluor TM 450 Pacific Blue TM Fixable Viability Dye efluor 505 9
Blue Laser Options Red Laser Options Green Laser Options Fluorochromes example for a 7 color stain using the red and blue laser line 57 2010 www.ebioscience.com How can we go further??? What about a 10 colour stain???? EMPOWER YOUR VIOLET LASER!!! The Next Generation in Fluorescence efluor Nanocrystals 10
efluor TM Nanocrystal Composition Semiconductor Nanocrystals; The principal Core Shell Lipid Layer Semiconducting Infinite Solid Conduction Band (LUMO) A high-energy photon excites an electron across the bandgap Antibonding orbital (mostlymetal d character) Bandgap (Eg) Core composition and sizedetermines color of the Nanocrystal Inorganic shell improves stability and brightness 2-10 nm CdSe ZnS Water Soluble Nanocrystal Unique, propriartity lipid coating provides water solubility and functional groups for conjugation Biomolecules arecovalently attached to functional groups on coated nanocrystal Bonding orbital (mostlyanion pcharacter) Semiconducting Nanocrystal Valence Band (HOMO) A bandgap-energy photon isemittedas theelectronfalls back tothegroundstage The excited complex is a very strong reductant photobleaching The shell keeps the excited electron from interacting with the environment Eg(0), α and β are material constants efluor Nanocrystal Platform Technologies efluor Nanocrystals Excitation/Emission Emission Comparison Fl. Microscopy Flow In Vivo Large Stokes Shift Narrow emission = minimal compensation Excellent photostability Compatible with conventional fluorochromes/ Filter sets The efluor Solution: Compensation Requirements Violet Laser Options efluor Solution Traditional 11
Fluorochromes example for a 10 color stain using the violet, red and blue laser line 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 Flow Cytometry: Experimental Design Design and analysis of multicolor flow cytometry experiments Design Instrument configuration Fluorochrome performance FluorPlan Spectra Viewer Tandem dye considerations Compensation considerations Analysis Eliminating false positives Proper controls for gating Instrument 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 using 8-peak beads Blue laser (488 nm) FITC: PE: PerCP-Cy5.5: PE-Cy7: Red laser (633 nm) APC: APC-eFluor 780: Violet laser (405 nm) efluor 450: efluor 605NC: 12
Fluorochrome 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 Index Stain Index (SI) = D/W D = difference between pos and neg peak medians W = 2 * robust SD Relative brightness of fluorochromes using CD4 Combining detector & fluorochrome performance: assessment of dim antigens 8-peak beads in: FITC detector PE-Cy7 detector APC-eFluor 780 detector efluor 450 detector PBMC stained for CD4: CD4 FITC CD4 PE-Cy7 CD4 APC-eFluor 780 CD4 efluor 450 Fluorescence Intensity (arbitrary units) 1200 1000 800 600 400 200 Optimizing instrument settings: PerCP-Cy5.5 Cy5.5 & PE-Cy7 example PerCP-Cy5.5 PE-Cy7 0 350 450 550 650 750 850 Emission Wavelength (nm) Optimizing instrument settings for B220 PerCP-Cy5.5 Cy5.5 & CD3 PE-Cy7 B220 PerCP-Cy5.5 single stain PerCP-Cy5.5 spillover in the PerCP-Cy5.5 detector PE-Cy7 detector 626 volts 669 volts 626 volts 520 volts CD3 PE-Cy7 669 volts 520 volts CD3 PE-Cy7 CD3 PE-Cy7 CD3 PE-Cy7 B220 PerCP-Cy5.5 80% B220 PerCP-Cy5.5 12% B220 PerCP-Cy5.5 13
Design and analysis of multicolor flow cytometry 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 FluorPlan to determine fluorochrome options 80 2011 www.ebioscience.com FluorPlan Spectra Viewer FluorPlan Spectra Viewer 2011 www.ebioscience.com Design and analysis of multicolor flow cytometry 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 cytometry experiments Tandem Dye Consideratons Loss of FRET efficiency Fixation use freshly prepared, high quality formaldehyde Light exposure Shelf life 14
Fixation & photostability of PE-Cy7 Fixed cells: 2% Formaldehyde 30 minutes Room temp PE detector Freshly stained & analyzed cells Fixation & photostability of APC-eFluor 780 Fixed cells: 2% Formaldehyde 30 minutes Room temp APC detector Freshly stained & analyzed cells Fixed cells: 2% Formaldehyde overnight 4⁰C Fixed cells: 2% Formaldehyde overnight 4⁰C Live cells: Ambient light 6 hours Room temp Live cells: Ambient light 6 hours Room temp Additional 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 cytometry 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 experiment 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 cytometry experiments Design Instrument configuration Fluorochrome performance FluorPlan Spectra Viewer Tandem dye considerations Nanocrystal considerations Compensation considerations Analysis Eliminating false positives Proper controls for gating 15
Analyzing a multicolor flow cytometry experiment 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 laser Principle of singlet gating Single-cell Doublet laser Signal intensity Signal intensity time time 92 2011 www.ebioscience.com Practice of singlet gating Value of singlet gating CD3+ CD19+ doublepositive cells? No! Just 2 cells stuck together. Dead cells interfere with optimal staining resolution Fresh thymocytes Eliminating false positives with singlet gating & exclusion of dead cells Singlets Viable Anti-CD3-stimulated thymocytes 2011 www.ebioscience.com 96 2011 www.ebioscience.com 16
Analyzing a multicolor flow cytometry experiment 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 phenomenon 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 COMPENSATION COMPENSATION 100 2010 www.ebioscience.com Isotype controls: Ubiquitous An Ab of the same isotype conjugated to the same fluorochrome Rat IL-2 IgG2a PE 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 Limitations 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 17
Multicolor 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 from A to Z Apoptosis, Necrosis, Viability, Vitality, Functionality... Apoptosis Apoptosis vs Necrosis Greek: apo from, ptosis falling One of the main types of ProgrammedCell Death(PCD) Multi-biochemical event (incl. cell to cell interaction) Leadingto cell death; save disposeof 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, Apoptosisis 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 105 106 Induction and Regulation of Apoptosis Apoptosis Inducers in Cell Biology Induction Cell repair (e.g. damage due ionizing radiation ) Infection (e.g. v irus) Cell stress (e.g. starvation) Homeody namics (homeostasis) Regualtion cell-cell interaction (e.g. haematopoesis) Control: Cell signals Hormones Grow th factors Cy tokines Nitric ox yde Actinomycin D Camphtothecin Cycloheximide Dexamethasone Etoposide 107 108 18
The Apoptosis Process Cell shrinkage Chromatin Condensation Membrane Blebbing Apoptotic Body (AB) Formation 109 110 Continued Blebbing Nuclear Collapse Apoptosis induced Cell function changes membrane potential Membrane Potential Transition Pore opening Phosphatidyl Serine (PS) Translocation JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanineiodide) JC-1 is a membrane permeable dye for flow cytometry and fluorescent microscopy. Selectivelyenters the mitochondriawhere it reversibly changescoloras membrane potentials......increase (over values of 80-100 mv).this property is due to the reversible formation of J-aggregatesupon membrane polarization. Excitation: 488 nm @ 59% Caspase Activity 498 nm @ 82% 592 nm @ 100% Emission (max): 530 nm (JC-1 monomer) 590 nm (J-aggregate) JC-1is both qualitative, in regards to the shift from green to orange fluorescence emission,..and.quantitative, as measured by fluorescence intensity, in both filter sets. Fragmentation Metabolic Activity Plasmamembrane Integrity Condensation 111 112 Apoptosis induced Cell function changes Cytochrome C detection Transition Pore opening Cytochrome C Release Membrane Potential Transition Pore opening Cytochrome C Release Membrane Potential Phosphatidyl Serine (PS) Translocation Caspase Activity Fragmentation Plasmamembrane Integrity Condensation Metabolic Activity Cytochrome C detectionvia Monoclonal Antibodies Human Mouse Rat Flow Cytometry Western Blot Immuno Histochemistry Imaging Immuno Precipitation Functional Assays ELISA Human Cytochrome c Platinum ELISA 113 114 19
Calcium Sensing Dyes Apoptosis induced Cell function changes 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(400to 300 nm) Peak emissionremains steady (~510nm) Peak excitation: depending on free Ca 2+ conc. between 300nmand 400nm Peak emission: 510 nm Molecular weight: 1001.86 Da Membrane Potential Transition Pore opening Phosphatidyl Serine (PS) PhosphatidylSerine(PS) Translocation Translocation Indo-1 AM singleexcitation/ dual emission dye UnboundIndo-1hasa peakemissionat485nm,whichshiftsto 410nm uponca 2+ binding.inflowcytometry,this shiftcanbemeasuredovertime and represented asa ratio of the two emission wavelengths. Peak Excitation: 346 nm Peak Emission: 475 nm (Unbound Indo-1 =485nm shift to 410nm dep. on Ca 2+ binding) Molecular Weight: 1009.91 Caspase Activity Calcium Sensor Dye efluor 514 Indicator for intracellular free calcium Detection by: FC, IHC, Imaging, Microplate Readers Increased cellular uptake increased brightness Excitation: 488nm Emission: 514nm Calcium binding affinity: Kd = 232 nm MW:~1100Da Not recommended for quantitative measurements CalciumSensor Dye efluor 514 Jurkat cells were harvested, washed and loaded with CalciumSensorDye efluor 514for30minutes at37 C. The left panel shows cells that were washed and analyzed by flow cytom etry unstimulated (blue histogram) or stimulated with 1 ug/mlionomycin(purplehistogram).the right panel shows Jurkat cells loaded with Calcium Sensor Dye efluor 514 that were acquired on a flow cytometer for1minute andthenremoved for theadditionof 1ug/mL ionomycin and immediately placed back on the flow cytometer for continued acquisition.. Fragmentation Plasmamembrane Integrity Condensation Metabolic Activity 115 116 Detection of Phosphatidyl serine (PS) translocation via Annexin V labeling Detection of Phosphatidyl serine (PS) translocation via Annexin V labeling Early Apoptosis Late Apoptosis Necrosis Cell Membrane Phosphatidyl serine Cytosol Control: Annexin V FITC & Propidium iodid Etopodise treated Thymocytes Untreated Treated Counterstain with cell impermeant -dye e.g. PI, 7AAD Formats@eBioscience: 10µM Camptothecin, 4h efluor 450 FITC R-PE PerCp-eFluor 710 APC PE-Cy7 Biotin MAG 40x MAG 40x MAG 100x 117 118 Apoptosis induced Cell function changes Caspase activity Transition Pore opening Membrane Potential Phosphatidyl Serine (PS) Translocation Caspase Activity Caspase Activity Caspase 1 (pc) H WB, IP, IHC Caspase 2L p12 H,M,R WB, IP, IHC Caspase 3 (pro) M WB Caspase 3 (pc) H WB, IP, IHC Caspase 7 H WB Caspase 8 H WB Caspase 10 H WB Caspase 11 M FC Caspase 12 H,M,R WB Caspase 12 M WB, IP, IHC, FC Caspase 13 H,M,R WB Fragmentation Plasmamembrane Integrity Condensation Metabolic Activity WB: IP: IHC: FC: Western Blotting Immuno Precipitation Immuno Histo Chemistry Flow Cytometry H: Human M: Mouse R: Rat 119 120 20
CaspGLOW Apoptosis induced Cell function changes 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 Simple Protocol Collect cells and resuspend in incubation buffer Add caspase probe and Incubate @37C for 20min Membrane Potential Transition Pore opening Phosphatidyl Serine (PS) Translocation Caspase Activity Analyze Fragmentation Metabolic Metabolic Activity Plasmamembrane Integrity Condensation Data provided by: 121 122 Metabolic activity (Viability dyes) Calceinis 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 esterasesthereby 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 Metabolic activity (Cell Tracking and Proliferation) Cell Tracking & Proliferation Dye CFSE [5-(and 6)-Carboxyfluoresceindiacetatesuccinimidylester] Cell tracking and proliferation studies Also used in CTL assays and cell motility studies CFSE readily crosses intact cell membranes, inside the cells, intracellular esterasescleave the..acetate groups to yield the fluorescent carboxyfluoresceinmolecule. The succinimidylester..group reacts with primary amines, crosslinking the dye to intracellular proteins. Cell division can be measured as successive halving of the fluorescence intensity of..... CFSE 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 123 124 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 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) 125 126 21
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 Simultaneous Measurement combining Proliferation Dyes: CFSE or Cell Proliferation Dyes Immunophenotyping; surface markers Live/Dead discrimination; 7AAD, FVD Simplified Protocoll Safes up to an 1h time compared to conventional methods Nuclear stains; BrdU, transkription factors Cytokine expression Annexin V The staining is always depended on the cell type. Fixation and Permeabilisation can effect performance. Some buffer might effect performance. 127 128 Apoptosis induced Cell function changes Plasma membrane integrity Transition Pore opening life/dead discrimination using cell impermeant dyes Membrane Potential Phosphatidyl Serine (PS) Translocation Caspase Activity Propidium Iodide (PI) Exclusion of nonviable cells in flow cytometric analysis Binds to double stranded 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. Mouse thymocytes were prepared as a single ce ll suspension and incubated overnight at 37 C in medium (left) or medium with 1µM dexamethasone (right). Cells were harvested and stained us ing the Annexin V FITC Apoptosis Detection Kit and Propidium Iodide Staining Solution (cat. 00-6990). 7-amino-actinomycin D (7-AAD) Fragmentation Plasmamembrane Integrity Condensation Metabolic Activity 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). 15% 7AAD 7AAD stain on human Lymphocytes after freeze/thaw cycle 129 130 Life/Dead discrimination; Fixed Cells Life/ Dead discrimination; Fixed Cells Amine-reactive fluorescent reagents Binds free amine groups on proteins Fixable Viability Dye efluor 450 Fixable Viability Dye efluor 506 Fixable Viability Dye efluor 660 Fixable Viability Dye efluor 780 Unlabeled cells NO fluorescence Fixable Viability Dyes are a viability dyes that can be used to irreversibly label dead cells prior to: Cryopreservation Fixation Permeabilization Unlike 7AADand 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 excludedfrom analysis when intracellular targets are being studied. Fixable Viability Dyes may be used to label cells from all species. Labeled cells Intact membrane Labeled cells Compromised membrane MEDIUM fluorescence BRIGHT fluorescence 131 132 22
Life/ Dead discrimination; Fixed Cells Nuclear Labeling of Amine-reactive fluorescent reagents Binds free amine groups on proteins FixableViabilityDyeeFluor 450 FixableViabilityDyeeFluor 506 FixableViabilityDyeeFluor 660 FixableViabilityDyeeFluor 780 NO fluorescence MEDIUM fluorescence BRIGHT fluorescence Cat. 65-0880 Nuclear Red (DRAQ5 ) Ex: 488-647nm Ex: 488nm Detect: PerCP-eFluor 710 or PerCP-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. anthraquinone dye high affinity for double-stranded membrane-permeable dye nuclear content analysis eg. for ploidy /cell cycle analysis because it binds stoichiometrically In fluorescent microscopy, it can be used as a nuclear counterstain. For cell cycle/ 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) 133 134 Nuclear Labeling of Cell Tracking; CellVue dye Cat. 65-0881 Nuclear ORANGE (CyTRAK Orange ) Ex: 488-550nm Ex: 488nm Em max: 610nm (610/20 BP) lipophilic dyes labeling of the cell membrane identifying and tracking of labeled cells rapid and stable labeling Multiplexable Supplied with diluent and labeling vehicle anthraquinone dye high affinity for double-stranded 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. CellVue Lavender (Cat.88-0873) Ex: 420 Emmax: 461 CellVue Jade(Cat.88-0876) Ex: 478 Emmax: 508 CellVue NIR780 (Cat.88-0875) Ex: 633 Emmax: 776 CellVue Maroon (Cat.88-0870) Ex: 647 Emmax: 667 CellVue Plum(Cat.88-0871) Ex: 652 Emmax: 671 CellVue Burgundy (Cat.88-0872) Ex:683 Emmax: 707 CellVue NIR815 (Cat.88-0874) Ex: 786 Emmax: 814 Please make sure that your instrument is capable of detecting this dye. 135 136 Apoptosis induced Cell function changes break vs total : APO-DIRECT Membrane Potential Transition Pore opening Phosphatidyl Serine (PS) Translocation Detection of apoptotic cells by flow cytometry 2-color staining method Labeling breaks and total cellular The kit content: Caspase Activity 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 Fragmentation Fragmentation Plasmamembrane Integrity Condensation Metabolic Activity One ofthe most easily measured features ofapoptotic cells is the break-up ofthe genomic by cellular nucleases. These fragments can be extracted from apoptotic cells and result in the appearance of laddering when the is analyzed by agarose gel electrophoresis. The of non-apoptotic cells that remains largely intact does not display this laddering on agarose gels during electrophoresis. The large number of fragments appearing in apoptotic cells results in a multitude of 3'-hydroxyl termini in the. This property can be used to identify apoptotic cells by labeling the 3'-hydroxyl ends with directly conjugated fluorescein- deoxyuridine triphosphate nucleotides (FITC-dUTP). The enzyme terminal deoxynucleotidyl transferase (TdT) catalyzes a template-independent addition of deoxyribonucleoside triphosphates to the 3'-hydroxyl ends of double- or single-stranded with either blunt, recessed or overhanging ends. A substantial number of these sites are available in apoptotic cells providing the basis for the method utilized in the APO-DIRECT Kit. Non-apoptotic cells do not incorporate significant amounts of the FITC-dUTP due to the lack of exposed 3'-hydroxyl ends. APO-DIRECT is a trademark of Phoenix Flow Systems, San Diego, California. 137 138 23
APOSTAIN Apoptosis induced Cell function changes Anti-ss stain of condensed chromatin Staining of cells and/or tissue sections with APOSTAIN followed by heat treatment, induces denaturation in situ only in..apoptotic nuclei. In the presence of formamide, only apoptotic nuclei becomes denatured and..detectable Applicable for Human, Mouse and Rat samples For Flow Cytometry For IHC staining including formalin fixe, paraffin embedded tissue sections Membrane Potential Transition Pore opening Phosphatidyl Serine (PS) Translocation Caspase Activity Fragmentation Metabolic Activity Plasmamembrane Integrity Condensation DAPI counterstain APOSTAIN positive nuclei 139 140 The ebioscience Knowledge Center The ebioscience Knowledge Center www.ebioscience.com www.ebioscience.com 141 142 The ebioscience Knowledge Center The ebioscience Knowledge Center www.ebioscience.com www.ebioscience.com 143 144 24
..perfection is finally attained not when there is no longer anything to add, but when there is no longer anything to take away... Thank You Antoine de Saint Exupéry (1900-1944), Terre des Hommes (1939) 145 146 25