Presentation on theme: "Applications in Flow Cytometry"— Presentation transcript:
1 Applications in Flow Cytometry some of the most common aplications of Flow CytometryVast subjectwon’t go into much detailmain objective is to give you a brief overviewexemplify what can be done with techniqueCláudia BispoIGC – April 4, 2013
2 Outline Potential Applications of Flow Cytometry Cell State ImmunophenotypingCell cycleApoptosisCell proliferationCell FunctionCell activationCalcium fluxCytokine SecretionActivation of signalling pathwaysLevels of intracellular reactive oxygen speciesoutline, during this presentation I will be talk about applicationsassess the state of the cellular populationsfurther study functional state.these applications can be used most samples,some minor optimization steps can be necessary for bacterial and other microbiological samplesfew techniques that can be useful to people from microbiologyMicrobiologyDead/Live DiscriminationAbsolute countingTime points
4 CAUTION – Abs selection: ImmunophenotypingUses labeled antibodies (Abs) to identify cells of interestDetermination of cell surface antigensAllows for detailed identification of cellular subsets (simultaneously measure multiple parameters cell by cell)Targets on both surface and intracellularlyimmunophenotyping, the most common of flow assays.uses labeled antibodies to identify cells of interest,with multiple detectors allowanalysis of multiple parameters cell by cell simultaneously,EXAMPLE FIGidentification of cellular subsets,labeled concurrently for different cellular markersit was possible to identifyJust as a footnote,important to know your instrument & select fluorochromesthat match the laser line and filter setup in the equipmentto prepare experiment correctly and not waste unnecessary reagentsCAUTION – Abs selection:Fluorophore’s excitation spectrum must match the laser line used, and its emission must fall within detection filter sets in the cytometer
5 Excitation / Emission : Cell CycleDNA content analysis - Propidium Iodide (PI)G2MG1SG0G0/G1G2/MS-phaseEvaluation of DNA content of cells - still one of the biggest applications in this field. can provide a great deal of information about the state of cells on your sample effect of certain stimuli, ex: transfected genes or drug treatment.This is achieved bylabeling cell nuclei with Propidium Iodide (PI)& then analyzing the fluorescence properties of cellular pop.As resting and G1 cells will have one copy of DNA,1x fluorescence intensity 1st peak to the leftCells in G2/M phase, two copies of DNA 2x the fluorescence intensity.Since the cells in S phase synthesizing DNA fluorescence values between 1st - 2nd populationsjust like we can see in this histogram.EXAMPLE FIG3 regions can subjectively selected percentage of cells in each phase.Fluorescence (DNA content)Excitation / Emission :488nm / max 617nm
6 Cell Cycle Analysis Cell Cycle Analysis Software Cell Number G0/G1Cell NumberExamples:FlowJoModFit LT FCS Express IDLYK… G2/MSHowever, often … is not the most correct. Useful if lot of debris or multiple DNA populations with different DNA contentto determine accurate nr of cells in each phasebest option use software that will mathematically analyse the DNA histogram& thus give a more accurate measurement of the percentage of cells in each phase.Fluorescence IntensityAccurate measurements allow for resolution of normal cells undergoing G1, S, G2 phasesAlso useful when multiple DNA populations present: measuring aneuploidy & polyploidy
7 Cell Cycle - Bromodeoxyuridine (BrdU) method Propidium Iodide plus BrdU staining• BrdU is thymidine analog• Taken up by cells in S-phase• Usually in combination with PI104S Phase103Anti-BrdU FITC102limitation of looking at a single fluorochrome: no kinetic informationif S-phase cells are actually cycling / synthesizing DNATo assess: use BrdU plus PI on the sampleBrdU is an analogue of thymidine will be taken up into the DNA of cycling cells After a specific amount of time, use an antibody against BrdU,find out if the cells took up BrdU during the process.EXAMPLE FIGplot, cells + for BrdUcells in S-phase region in the PI histogram,confirmed really cycling, excluding a few events that were not101G1G2/MExcitation / Emission :PI 488nm / max 617nmBrdU varies by fluorophorePropidium Iodide
8 Pyronin Y plus Hoechst 33342/33258 Cell Cycle - G0/G1 discriminationPyronin Y plus Hoechst 33342/33258G0/G1SG2/MG0G1Cell CountRNA ContentRNA contentFurthmore:to determine the level of senescence or resting stateoption to analyse cell cycle with dye combinationHoechst and Pyronin Ywhen cells are stained first with Hoechst and then with Pyroninpossible to distinguish DNA from RNA.Pyronin binds preferentially to RNA& Hoechst to A-T DNA base pairsb/c resting cells arrested in G0 phase, have lower level of RNAand so are in red region of plot or bottom scheme G0 and G1 phases can be separated looking at differences in RNA contentDNA content (A-T base pairs)Excitation / Emission :PY 488nm / 575nmHO UV line / nm
9 Apoptosis Changes in light scatter DNA denaturation apoptotic cells can be recognized bycharacteristic pattern of morphological, biochemical and molecular changes, summarized:Flow cytometry use some of these detect and identify apoptotic cellsmajority based on the measurement of light scatter,sensitivity of DNA to denaturation,detection of changes in plasma memb or in signaling pathways.positive controlgive useful information about what to expect from analysisb/c the basal level of apoptosis and necrosis varies a lot in cellular population and might not give the necessary infocells in which apoptosis has been induced by (for example, by fas induction, treatment with the topoisomerase I inhibitor, etc…)DNA denaturationChanges in plasma membraneChanges in cell organelles / signaling pathways* positive control is useful
10 Apoptosis CELL DEATH – FSC x SSC Changes in light scatter: Regarding changes in light scatter,as cells die/become apoptoticrefractive index of internal cytoplasm becomes similar to that of the extracellular mediumreduction FSC signal, intracellular changes & alterations of cytoplasmic memb increase SSC EXAMPLE FIGcells cultured with rapamycin, an apoptosis inducer OR activation inhibitor% of cells in region decreasesbutother ex. changes is morphology might allow for an easy discriminationadding viability dye proper differentiation live/deadwith light scatterlow level resolution of dead and apoptotic cellsin some cases not enoughChanges in light scatter:low level resolution of apoptotic cells
11 Propidium Iodide (fixed cells) ApoptosisPropidium Iodide (fixed cells)DNA Degradationseveral viability dyes, stain dead cells,looking into DNA content in fixed samplesduring apoptosis, DNA is degraded within the DNA there are nicks and fragmentation.looking at DNA content with PIcells that have lost DNA will take up less stain and so appear to the left of the G1 peak amount of cells on SubG1 peakother viability dyes are availabledepend on the experiment:stain other fluorochromes,if you plan to fix and permeabilize your cells, etc.This method relies on the fact that after DNA fragmentation, there are small fragments of DNA that are able to be eluted following washing of the sample.This means that after staining with a quantitative DNA-binding dye, like PI, cells that have lost DNA will take up less stainand like we can see in this histogram, appear to the left of the G1 peak.Other viability dyes :7-AADZombie AquaTo‐Pro3...
12 Annexin V-fluorochrome plus Propidium Iodide (non-fixed cells) ApoptosisAnnexin V-fluorochrome plus Propidium Iodide (non-fixed cells)To assess cell apoptosis in live cells, apoptotic/necrotic cells:Annexin V + PI.Annexin-V is a specific phosphatidylserine-binding protein that can be used to detect apoptotic cells.In normal cells this residues found in inner part of cytoplasmic memb.during apoptosis translocated & externalised Annexin V is able to bind to themIn general, this is an early event in apoptosisb/c cells are not fixed we can further exclude dead cells using PI---Annexin-V is available conjugated to a number of different fluorochromes.
13 Annexin V plus Propidium Iodide ApoptosisAnnexin V plus Propidium Iodidethus, distinguish four populations:• Viable cells, not undergoing detectable apoptosis:Annexin V (–) and PI (–)• Early apoptotic cells:Annexin V (+) and PI(–)• Late apoptotic cells:Annexin V (+) and PI (+)• Cells that have died through non-apoptotic pathway:Annexin V (–) and PI (+)commonly: PI in their samples but as you can see only these 2 pop will be identifiedExcitation / Emission :Annexin varies by fluorophorePI 488nm / 617nm
14 Analysis by Flow Cytometry Apoptosis (intracellular staining)Fix and permeabilizeAdd AntibodyAnalysis by Flow CytometryIn addition to all these methods, intracellular staining for specific signaling pathways involved in apoptosishave also become a widespread practice in flow cytometrygeneral protocol for this type of analysis are…
15 Apoptosis – Bcl-2 family members evaluate apoptotic states through Bcl-2 expression,protein blocks apoptotic deathJust like as we can see in the EXAMPLES when cells are stimulated & have higher cell viability the expression levels of this protein are higherwhen apoptosis is induced (rapamycin) mean expression levels decrease correspondelycorrelate effectsBcl-2 proteins block apoptotic deathby controlling mitochondrial memb permeability & interfering with pro-apoptotic proteins.Excitation / Emission:Varies by fluorophore
16 Apoptosis – Activated forms of Caspases UntreatedEtoposidefundamental aspect activation of caspases,many available conjugated Ab to bind to the active forms of caspases.EXAMPLEuse cleaved Caspase-3 Antibody (Alexa Fluor 488 conjugate)clear difference between the expresssion during normal state low expression levelswhen a cytotoxic agent is added expression of active form of caspase is greatly incresasedFlow cytometric analysis of Jurkat cells, untreated (blue) or etoposide-treated (green), using Cleaved Caspase-3 (Asp175) Antibody (Alexa Fluor® 488 Conjugate).Excitation / Emission:Varies by fluorophoreEx A488: 488nm / 520nm
17 Cell ProliferationTracking Cell Proliferation with CFSE (Carboxyfluorescein Succinimidyl ester)Dilution of CFSECell DivisionsSTAIN WITH CFSECELL1234Evaluate cell proliferation,use of CFSE allows to track cell proliferationthrough- diffusion of this fluorescein molecule- freely into cells- Retained in their cytoplasm,- not affecting its cellular functioncells labelled with this dye proliferate,after mitosis each daughter cell: contain half as much dye as the parent half as bright.After each division thelevel of fluorescence will halve by measurement of the green fluorescence monitor the nr of divisions.EXAMPLE,cells have been labelled & stimulated - follow divisions up to 5 generations. Original + daughter cellsAlso, combining surface markers: specific subpopulations, like in this case with B cells.Using a modelling program such as FlowJo or ModFit,further determine how many cells belong to each generation.Excitation / Emission:488nm / 521 nm
18 Tracking Cell Proliferation with CFSE IL-7IL-7+ DMSOIL-7+ PI3K InhibitorIL-7+ Erk Inhibitorfew other examples when the cells are stimulatedor inhibited to proliferate
20 Cell Activation FSC x SSC – Cell size similar to apoptotic cells, also possible to evaluatethe changes in morphology of activated cellsWhen cells are activated bigger cellular population with higher FSC and SSC distribution.through light scatterwith a low level resolutionSofia Marques (IMM)Filipa Lopes (IMM)
21 Cell Activation Activation markers: CD69, CD71, etc more detailed and specific way to assess cellular activationspecific expression of certain activation markers, just like CD69 or CD71 …to assess effect of stimulilarge range of antibodies to choose from and combined with others det. activated state specific subpopExcitation / Emission:Varies by fluorophore
22 Calcium Flux Excitation / Emission: Indo1 UV line / 405nm Effects of T cell receptor stimulation by B-Cells on ionized calcium concentration ([Ca2+]i).Fluorescence-imaging of human erythrocytes treated with PGE2 using the calcium fluorophor Fluo-4Calcium very important cell signalchanges in intracellular levels are a good indicator if the cell is responding to a stimulus.mobilization observed using fluorescent dyesThe most common UV-excited dye Indo-1 fluoresces at a different wavelength when bound to calcium & when it is not.By measuring fluorescence at both wavelengths ratioing the two signals, a flux can be seen THROUGH course of 20minFluo-4 is a excited by a 488 laser when binds to Ca2+ inside the cell fluorescent---Calcium ions have an important role in cell signalling and the intracellular concentration of calcium ions may show transient changes in response to external stimuli. There are several fluorescent dyes whose properties depend on the amount of bound Ca++. For flow cytometry, the most useful of these is indo-1 whose emission wavelength changes on binding calcium (see Figure 3.4, Chapter 3). It is loaded into the cell in the form of the acetoxymethyl ester.Indo-1 is excited in the UV. A mixture of fluo-3 and Fura red can be used for work with a flow cytometer limited to excitation at 488 nm. Fluo-3 has a maximal fluorescence at 530 nm; its fluorescence increases with increasing Ca++ concentration. Fura Red has a maximal fluorescence at 650 nm which decreases with increasing Ca++ concentration.Excitation / Emission:Indo1 UV line / 405nmFluo nm / 516nmSofia Marques (IMM)Filipa Lopes (IMM)
23 Activation of signalling pathways Evaluation of the activation signaling pathwaysanother way to assess cellular responses to environmental stimulicritical decision processes within the cellcombining phospho-specific antibodies & flow cytometry tool for detection of protein phosphorylation at the single cell level.General phospho-protein staining technique:after certain stimulicells are then fixed, permeabilized & stained with phospho-specific antibodies.b/c the Abs bind only to the phosphorylated form of the proteins increase in fluorescence correlates with an increase in phosphorylation.stimulus A increase in red fluorescence because the red protein is phosphorylated.B green FLThe combination of stimuli A and B induces phosphorylation of both proteinsmaking the cells both green and red fluorescent, that isdouble positive for both dyes.Krutzik et al. Clin Immunology (2004)
24 Phospho-protein detection Activation of signalling pathwaysPhospho-protein detectionpStat1Multiple signaling cascades can be monitored simultaneously:different Abs determine the specificity of stimulusanalyze rare subsets of cells within complex populationsBetter discrimination of High vs. Low respondersBetter evaluate population heterogeneityimprovement comparing with Western Blot:clearly 2 different kinds of samples appear identical evenreliably distinguish different levels of expressionAbility to analyze rare subsets of cells within complex populationsBetter evaluate population heterogeneityDiscrimination of High vs. Low respondersKrutzik et al. Clin Immunology (2004)
25 Combining Surface Markers with Phospho-staining Another advantage of using flow cytometryincorporating surface markers into staining protocolsdifferent sub pops analyzed concurrentlyfor protein phosphorylationmaximize results from precious samples… Evaluate protein phosphorylation in different subpopulations concurrently
26 Levels of intracellular reactive oxygen species Excitation / Emission:488nm / 605nmother type of evaluation isanalyze the oxidative burst after stimulusseveral compounds, in their reduced form are non-fluorescent become fluorescent upon oxidation.ex: dihydroethidium,when oxidized to ethidium bind to DNA of cells and fluoresce red.incubation with 10 mM muramyl dipeptide for 15 min.(small window of time – time points useful to not miss)Measurement of oxidative burst in human peripheral blood granulocytes using dihydroethidium. On oxidation, ethidium is produced which binds to DNA & fluoresces red.The histogram shows the red fluorescence before and after incubation with 10 mM muramyl dipeptide for 15 min.
27 Cytokine Secretion - Multiplex Bead Arrays SOON@ UICLuminex MAGPix Systembead coated with capture antibody mixtureQuantitation & detection of cytokine and signal transduction proteins, DNA & RNA- Magnetic bead-based technology- Up to 50 analytes /well of a 96 well plate- Up to results in 1 hour- Uses small sample volumesLuminex System analyzer based on the principles of flow cytometrymagnetic bead-based technology multiplex up to 50 analytes in a single microplate well (very small sample volumes)system uses differentially dyed beads to achieve multi-analyte profilingfor proteins & nucleic acids.Each type of bead coated with specific antibody mixture specific for analytes measured,These beads then pass through the equipment flow cell,b/c they are magnetic they’re stop and optics able to measureamount of fluorescencebeing proportional to amount of cytokine bound to each bead(equivalent to ELISA reading)but more cost and time efficient
28 Multiplex Bead Arrays SOON @ UIC Or design custom and personalized panelsMerck MilliporeMAGPixA wide range of molecules, such as cytokines, hormones, so on… can be analyzed andMany panels are available, examples Merk Milliporepossibility to design your own panels for the molecules you’re interested inThe procedure is really straight forward and you’ll be able to get up to results in 1 hourgraph;:
29 Microbiology Applications Like I saidMost these applications can also be applied to bacterial and other microbiological samplesFew techniques that can be useful specifically to people from microbiology
30 Microbiology - Dead/Live Staining Various established methologies can be optimizedSYTO 9 /PI - Live/Dead BacLight viability kitSYBR Green-I /PI Barbesti (2000)DiBAC4(3)/EB/PI Flow Cytometry Protocols 2nd ed.etc...Several kits availableExcitation / Emission:488nm / Varies by methodLIVE/DEAD kit for BacteriaLIVE/DEAD kit for Yeast & Fungilive dead staining various established methologies can be optimized for bacterial and other microbiological samples.encoutered several already published optimizationsand several kits are also commercially available.One thing to account for is the excitation/emission spectra of these do not coincidewith fluorescent tags of your sampleLIVE/DEAD BacLight Bacterial Viability KitSYTO 9 stain + PIThese differ in their ability to penetrate healthy bacterial cells.alone, SYTO9 labels live and dead bacteria.PI only bacteria with damaged membranes,SYBR-I + PIto detect viable bacteria in different samplesSYBR-I (+) PI (-): viable bacteria;SYBR-I (-) PI (+): dead or damaged cells,PI (+), while SYBR-I (+): partially damaged membranes, the PI does not reach a concentration that is sufficient for an efficient energy transfer from SYBR-I to the PI bound to DNA.DiBAC4/EB/PIDiBAC4 which is used as an indicator of memb potential ΔΨ, with EB, which is retained by cells with intact membranesPI is used to demonstrate membrane permeability; once PI enters cells, it displaces EB from nucleic acidsCAUTION:Make sure excitation/emission of kit dyes do not coincide with the ones on your sample
31 Microbiology Monitoring Cell Cycle – DNA content Minor protocol adjustments/optimization might be needed comparatively with established protocols for eukaryotesE.coli: DRAQ5 - Silva et al. (2010) Yeast : PI or SYTOX Green - Knutsen (2011)There are also few papers published on how to evaluate DNA content for microbiological samplesDRAQ5 with E.coliPI or SYTOX Green with yeastExcitation / Emission:647nm / 670nmExcitation / Emission:488nm / 520nm
32 Microbiology Time Points Assess treatment effect on population survival, protein expression, etc at indicated time points- effect on protein expression- counting: population proportionsCAUTION:Make sure you use a fluorescence control when protocol requires measuring & comparing protein expressionTime points:assess treatment effect, great importance no matter what is your research background.population survival, cell cycle, etc during determinated time points,Or the effect on protein expressionIn this last case it is of maximum importance use a fluorescence control,such as fluorescent beads at least at the beggining of each analysis.eliminates de effect of several factors such as: PMT ou laser differences from day to day,facilitating the comparison of results counting cells and assess population proportions
33 Cell counting Beads @ UIC Scepter Employs the use of reference beads on a regular cytometerAllows the examination of large number of cells per sampleCombining Surface Markers will allow to count specific subpopulations@ UICScepterHandheld Automated Cell CounterAllows cell differentiation by its volume (size) according to the Coulter principleUses a disposable sensorto count cells on a regular cytometer you must use reference beadsb/c most flow cytometers have no way of exactly controlling the flux and amount of sample,examine of a large number of cellsreduce the time employed with each determination: for large-scale studies this may be ideal.possibility of combining surface markers during the processallow for determinations of specific subpop.The easiest way to accomplish this is reference beadswith known number of particles per mL can be added to the sample.There are other alternatives:Scepter: handheld cell counterdifferentiates cells by its volumeUses disposable sensorCoulter principleThe Coulter method of sizing and counting particles is based on measurable changes in electrical impedance produced by nonconductive particles suspended in an electrolyte.A small opening (aperture) between electrodes is the sensing zone through which suspended particles pass. In the sensing zone each particle displaces its own volume of electrolyte. Volume displaced is measured as a voltage pulse; the height of each pulse being proportional to the volume of the particle.The quantity of suspension drawn through the aperture is precisely controlled to allow the system to count and size particles for an exact reproducible volume.Some disadvantages using beads for counts. Most importantly:1.Beads tend to aggregate2.bead concentration may vary(depends on bead aggregation, how well you vortex the beads, whether or not evaporation has occurred in the bead stock, pipetting issues)
34 Image based alternatives Cell counting (2)Countess™ Automated Cell Counter (Invitrogen)Tali™ Image-Based Cytometer (Invitrogen)TC20 Automated Cell Counter (BioRad)etc...Image based alternatives@ UICSOON@ UICImage based alternative:-Countess (we have at UIC)-TC20 from bio-rad-TaliAre automated cell counters,provide the total cell count & assess cell viability via trypan blue exclusionIn case of Tali & Muse (son deliver)more complex systemActually MINI cytometer,Evaluate GFP/RPF expressionRun CELL CYCLE, Anexin and other apoptis assays, among otherlike the others,Easy to work with,Get comprehensive data with graphic reportsright from your benchCoulter principleThe Coulter method of sizing and counting particles is based on measurable changes in electrical impedance produced by nonconductive particles suspended in an electrolyte.A small opening (aperture) between electrodes is the sensing zone through which suspended particles pass. In the sensing zone each particle displaces its own volume of electrolyte. Volume displaced is measured as a voltage pulse; the height of each pulse being proportional to the volume of the particle.The quantity of suspension drawn through the aperture is precisely controlled to allow the system to count and size particles for an exact reproducible volume.
35 Today’s Future Applications Field of Flow cytometry always progressing Not just in terms of reagents / fluochromes etcAlso new technologys & equipments
36 Amnis Image Stream Merck Millipore The ImageStream and FlowSight from Amnis (bought by Merck)are flow cytometers that combine flow cytometry with microscopythey take fluorescent pictures of every cell that runs through the instrument.resolution obviously not of confocal qualitybut can provide very useful information aboutCo-localization studies, cell cycle and so on...just a few examples:-regular plots-select pop. And then visualize itIn addition to the cell cycle analysis based on DNA content, image analysis features can be used to distinguish mitotic events from G2 cells based on nuclear morphology.
37 Amnis Image Streampower of this technology has in the last years been confirmed by the increasing number of applications being published,including co-localization studies,morphology analysis,cell cycle and mitosis,DNA damage and repair,cell-cell interaction,etc...
38 CyTOF – Mass CytometerCan measure 34 parameters simultaneously in a single cellCells are labeled with Abs containing rare elements metal tagsCells are atomized & ionized in plasma (high temperatures)Tags are separated & identified by mass (time-of-flight)The CyTOF instrumentbased on atomic mass spectrometry uniquely applied to single cell analysis.Cells are labeled with antibodies containing metal tags,introduced individually into the system, just like regular flow cytometeratomized & ionized with high temperatures plasmaBy time-of-flight is possible to separate by mass and counted.It can measure 34 parameters simultaneously in a single cellpresence of the tag element indicates that the antibody found and bound the target biomarker,and the intensity of the signal is directly proportional to the nr of Abs bound per cell.It’s stil a new technologya lot of optimizations are needed, still undergoingimense potential specially for evaluation and modeling of molecular networks at single cell levelIt is important to note that cells without any tagging elements cannot be detected by mass cytometry:Only element tags can be registered with high sensitivity and specificity, and therefore there is no “auto-fluorescence”-like effects.Elemental tags are chosen from rare elements whose natural concentration in a biological sample is below the detection limit.Unstained cells are “transparent” to the mass cytometer.Omatsky et al. JAAS (2008), Bendall et al. (2011)
39 Future Advances Heading further into the path of Single Cell Analysis microfluidics & lab-on-a-chip systemsReduced laser size and capillary flow techniques meansmaller instrumentsInstruments can now image cell at point of laser interrogationMore colours for immunofluorescenceWith the recent advances in flow cytometrywe are heading further into the path of single cell analysis-many advances have been made in microfluidics and lab-on-a-chip systems;-there are now instruments (Amnis) that can image a cell at the point of laser interrogation;And there is now the-measure at least 34 parameters simultaneously with CyTOF.This is indeed area constantly evolvingWith still a lot exciting techniques & possibilities to be discoveredHope I was able to pass along that messageand maybe interested you into looking forward to test some of these applicationsany help we can give isalso an oportunity for us to gain more experience in Flow CytometrySo don’t be afraid to ask!