3The Many Parts of Flow Experimental design Sample preparation Choosing the proper instrumentSetting up the instrumentCollecting the proper dataInterpreting the dataGraphics presentation and publicationSortingFlow BasicsData AnalysisSpecific Applications Courses
4Cytometry v. Flow Cytometry Localization of antigen is possiblePoor enumeration of cell subtypesLimiting number of simultaneous measurementsNo localization of antigensEnumeration of subtypes via cell/size/granularity/ markersCan look at numerous parameters simultaneously at high rate
5Uses of Flow Cytometry It can be used for… ImmunophenotypingDNA cell cycle/tumor ploidyMembrane potentialIon fluxCell viabilityIntracellular protein stainingpH changesCell tracking and proliferationSortingRedox stateChromatin structureTotal proteinLipidsSurface chargeMembrane fusion/runoverEnzyme activityOxidative metabolismSulfhydryl groups/glutathioneDNA synthesisDNA degradationGene expressionThe use of flow in research has boomed since the mid-1980s
6Mechanics of a Flow Cytometer Cells in suspension are brought in single file pasta focused laser where they scatter light and emit fluorescence that is filtered and collectedthen converted to digitized values that are stored in a file for analysisFluidicsOpticsElectronics
9Fluidics- Sample Differential 10 psi10.2 psi10.4 psi10.8 psiDifference in pressure between sample and sheathThis will control sample volume flow rateThe greater the differential, the wider the sample core.If differential is too large, cells will no longer line up single fileResults in wider CV’s and increase in multiple cells passing through the laser at once » » » Faster cell analysis BUT loss of resolution!!
10Effect of Flow Rate on Sample Data Resolution Low pressureHigh pressureEffect of Flow Rate on Sample Data ResolutionWith increased sample pressure, flow rate increases resulting in a decrease in data resolution.
11Optics- Light ScatterWhen light from a laser interrogates a cell, that cell scatters light in all directions.The scattered light can travel from the interrogation point down a path to a detector.
12Optics- Forward Scatter FSC DetectorLaser BeamOriginal from Purdue University Cytometry Laboratories
13Interrogation- Forward Scatter Light that is scattered in the forward direction (along the same axis the laser is traveling) is detected in the Forward Scatter Channel.The intensity of this signal has been attributed to cell size, refractive index (membrane permeability)Forward Scatter=FSC=FALS=LALS
14FSC DetectorCollectionLensSSCDetectorLaser BeamOriginal from Purdue University Cytometry LaboratoriesOptics- Side ScatterLaser light that is scattered at 90 degrees to the axis of the laser path is detected in the Side Scatter Channel. The intensity of this signal is proportional to the amount of cytosolic structure in the cell (eg. granules, cell inclusions, etc.)– Side Scatter=SSC=RALS=90° Scatter
15Why Look at FSC v. SSC Granulocytes Lymphocytes Monocytes RBCs, Debris,Dead CellsWhy Look at FSC v. SSCSince FSC ≈ size and SSC ≈ internal structure, a correlated measurement between them can allow for differentiation of cell types in a heterogeneous cell population
16Optics- Fluorescence Channels As the laser interrogates the cell, fluorochromes on/in the cell (extrinsic or intrinsic) may absorb some of the light and become excitedFluorochromes leave their excited state and release energy in the form of a photon with a specific wavelength, longer than the excitation wavelengthEmitted photons pass through the collection lens and are split and steered down specific channels with the use of filters.
17Optics- DetectorsThere are two main types of photo detectors used in flow cytometryPhotodiodesUsed for strong signals, when saturation is a potential problem (eg. FSC detector)Photomultiplier tubes (PMT)More sensitive than a Photodiode, a PMT is used for detecting small amounts of fluorescence emitted from fluorochromes.
18Optics- Fluorescence Detectors FSC DetectorCollectionLensLaser BeamFluorescenceDetector A, B, C, etc…Original from Purdue University Cytometry Laboratories, Modified by Benjamin Chojnacki
19Optics- FiltersDifferent wavelengths of light are scattered simultaneously from a cellNeed to split the light into its specific wavelengths in order to measure and quantify them independently. This is done with filtersOptical filters are designed such that they absorb or reflect some wavelengths of light, while transmitting others3 types of filtersLong PassShort PassBand PassDichroic
20Optics- Long Pass Filters Transmit all wavelengths greater than specified wavelengthExample: 500LP will transmit all wavelengths greater than 500nm400nm500nm600nm700nmTransmittanceOriginal from Cytomation Training Manual
21Optics- Short Pass Filter Transmits all wavelengths less than specified wavelengthExample: 600SP will transmit all wavelengths less than 600nm.400nm500nm600nm700nmTransmittanceOriginal from Cytomation Training Manual
22Optics- Band Pass Filter Transmits a specific band of wavelengthsExample: 550/20BP Filter will transmit wavelengths of light between 540nm and 560nm (550/20 = 550+/-10, not 550+/-20)400nm500nm600nm700nmTransmittanceOriginal from Cytomation Training Manual
23Optics- Dichroic Filters Long pass or short pass filtersPlaced at a 45º angle of incidencePart of the light reflected at 90º , and part of the light is transmitted and continues on.Dichroic FilterDetector 1Detector 2.
24Spectra of Common Fluorochromes Common fluorochromes used for violet, blue, and red laser flow cytometryFrom BD Multicolor Fluorochrome Reference Chart
25Electronics- Photovoltaics Once the detectors collect photons of light, they convert them to currentA voltage pulse is created as cells pass through the laserAs the cell passes into the laser, an event window opensMore light is scattered as the cell moves into the center of the laser (maxima)As the cell leaves the laser, less and less light is scatteredAfter a set amount of time, the window closes until another object enters the beamThe current is processed into signal that is then converted into a digitized value
26Electronics- The Pulse Time Photons/Detector (V)
27Digitization of the Pulse Pulse HeightPulse WidthPulse AreaTimeVoltage IntensityDigitization of the PulseEach pulse has a height (maximum voltage) and area. Area scaling can be performed to assure that data is linear, allowing comparisons to be made between data sets
28Electronics- Linear and Log Amplifiers When the current exits the detector, it passes through either a linear or log amplifier where it is converting it into the voltage pulse.Intensity of the voltage can be adjusted by amplifying it on a linear scale or converting it to a logarithmic scaleThe use of a log amp is beneficial when there is a broad range of fluorescence as that may need to be compressed (this is generally true of most biological distributions)Linear amplification is used when there is not such a broad range of signals (e.g. in DNA analysis and calcium flux measurement)
30Calibur PlatformThe BD FACSCalibur is a modular flow cytometer designed for cell analysis2 lasers488 (primary); detects FSC, SSC, and fluorescence633 (secondary); detects fluorescenceVarious protocols can be used for cell and DNA analysisImmuno-phenotyping (CD4, CD8)Apoptosis (PI and Annexin)Cell CycleGFP analysisHigh-throughput drug screeningFlexible system that is very easy to use and is very useful
31Calibur Platform- Fluorescence Detectors 488 Laser (Blue) has 3 parameters/filters to choose fromFL-1; 530/30FL-2; 585/42FL-4; 661/16635 Laser (Red) has 1 parameter/filterFL-3; 670LP
32FACSCalibur- Start-Up Fluidics should be started firstMake sure there is an appropriate amount of sheath fluid and that the waste container is not fullAlarms will sound if there is a problem with either container during startupPress the green button in the middle-right of the fluidics cart beneath the FACSCalibur bench to start pumpFlow cytometer should be started secondPress the green button on the right side of the Calibur to turn on the machineThe computer should always be started lastIf the computer is already on, be sure to restartLogin: AdministratorPassword: BDISAlways allow the instrument 15 minutes to warm-up prior to use
33BD CellQuest ProOnce logon is complete, find the menu icons displayed at the bottom-center and open BD CellQuest ProA new blank document will openGo to Acquire> Connect to CytometerConnects the instrument to the computerGo to Cytometer> Detectors and Amps, then return to the Cytometer menu and choose Compensation, Threshold, and StatusThese main palettes allow you to control the instrumentThe status palette will display if the instrument is ready for use
34BD CellQuest- Quality Control Once the instrument has warmed up and ready for use, a QC procedure called “Time Delay Calibration” must be performedAllows the flow cytometer to capture the signals at the right time from each laser as the cell passes through them in successionShould be performed every time a user turns on the machineGo to File>Open Document browse to Data 1>Setup FolderChoose Time Delay CalibrationGo to Cytometer>Instrument Settings, browse to Data 1>Setup folderChoose IS Time Delay CalibrationChoose Set, then DoneThis will load instrument settings for the calibrationLoad calibration beads onto the bulk injection port and press Run on the front of the cytometerClick Acquire, then go to CytometerSelect OK when message appearsA sound will acknowledge that the operation was performed successfullyUnload the calibration beadsPress the Standby button on the front of the cytometerThe cytometer is now ready for use (HUZZAH, etc.)!!!
35BD CellQuest- Creating a New Protocol If using a premade template, close the window that opened when CellQuest launchedIf the window was closed inadvertently, go to File>New DocumentIn order to acquire data, plots need to be createdGo to Plot and choose the appropriate typeGo to Windows >InspectorThis controls the formatting of the plots
36Creating a New Protocol- Plot Types There are several plot choices depending upon the experiment’s application and data being capturedSingle Color HistogramFluorescence intensity (FI) versus countTwo Color Dot PlotFI of parameter 1 versus FI of Parameter 2Two Color Contour PlotFI of P1 versus FI of P2. Concentric rings form around populations. The more dense the population, the closer the rings are to each otherTwo Color Density PlotFI of P1 versus FI of P2. Areas of higher density will have a different color than other areas
37Creating a New Protocol- Plot Types Contour PlotDensity PlotGreyscale DensityDot PlotHistogram
38BD CellQuest- Creating a New Protocol Once a plot type is selected, it will need to be formatted properlyWith the plot highlighted choose Acquisition to Analysis from Plot Type in the InspectorChoose the appropriate parameters (or parameter for histogram plots) for the axesRepeat until there are an appropriate number of plots for the analysisThis will be based on the needs of each projectPlace gates in each plot to distinguish areas of interestSince there is no data, gates can be placed in the plot where data is expected to beGates can be adjusted during data acquisition
39Creating a New Protocol- Gating Gates are used to isolate subsets of cells or “populations” on a plotAllows the ability to look at parameters specific to only that subsetCan use boolean logic to include or exclude multiple gatesFor scatter plots there are 3 main types of gates:RectangularGenerally used to define a region of interest; can also be used to define a broad population with outliersPolygonalUsed define clustered/dense populations of interestQuadrantMost often used in multicolor to distinguish negative populations from single- and double-positive onesFor histograms bar/horizontal gates are used to define spikes/peaks of interest
40BD CellQuest- Creating a New Protocol Once gates have been set around areas of interest, stats can be displayedHighlight a plot and choosing Histogram or Quadrant (depends on the plot type)Stat boxes can be edited to display any number of items pertinent to the experiment/projectStatistics can also be displayed for a plot without gates if neededOnce the plots are in place, go to File>Save Document AsNavigate to the appropriate investigator’s folderGive the protocol an appropriate name
41Creating a New Protocol- Naming Folders When performing analysis a new folder should be made to contain the data from that runGo to Acquire>Parameter DescriptionPalette allows control of instrument, naming of folders and sample (Sample ID)On the Acquisition Palette go to the Directory line and choose ChangeIn the navigation window go to Data 1>Sample Files C1>(PI’s Folder)Choose New FolderThe new folder should be named to include:Date (including year)Your InitialsA new folder should be made for each day of use
42Creating a New Protocol- Naming Files The default name for each file can be changed if more specification is neededOn the Acquisition Palette go to File line and choose ChangeIn the Custom Prefix line specify the name the files should haveIn the File line enter the desired suffix number of the first fileThis number will be incremented with each sample as data is savedEverything is prepared for acquiring data (HUZZAH, etc.)!!!
43BD CellQuest- Controls Every experiment performed on the cytometer should include controlsJust plain, good ol’ fashioned scienceDistinguish cell populations from debris or backgroundAllow differentiation of labeled cells (i.e., cell subtypes)Necessary controls will depend on the experimentCommon controls includeSingle negative controlsUnlabeled cellsIsotype controlsFluorescence minus one (FMO)Single positive controlsOne for each color being usedBD Comp Beads
44BD CellQuest- Setting Up Controls Start acquiring data with Negative or Isotype controlsIn the Acquisition palette check the Setup box next to the acquisition controlsPlace the control tube on the bulk injection portPress the Run button on the front of the cytometer and click AcquireAdjust the voltage of the appropriate parametersIf there is debris, adjust the thresholdData should placed so that it is easily viewable without going beyond the 2nd decade
45Setting Up Controls- Threshold When the laser interrogates an object, light is scattered.If the amount of light scattered surpasses a threshold, then the electronics opens a set window of time for signal detectionThe threshold can be set on any parameter, but is usually set on FSCThis is generally used to eliminate debris or cell fragments that are not of interest
46Setting Up Controls- Threshold FSCDetectorTimeThreshold(eg. 52)
47BD CellQuest- Setting Up Controls After the FSC, SSC, and threshold are adequately set, abort acquisition and uncheck the Setup boxBegin acquiring dataData will be automatically saved based on the settings performedOnce negative controls have been recorded, repeat the process with single positive controls or FMOsWhile running in Setup mode, adjust the parameter voltages to perform compensation
48Setting Up Controls- Cell Populations When adjusted correctly, FSC and SSC should differentiate cell populationsGranulocytesMonocytesLymphocytesRed Blood Cells/DebrisBased on granularity and size
49Setting Up Controls- Compensation Fluorochromes typically fluoresce over a large part of the spectrum (100nm or more)Depending on filter arrangement, a detector may see some fluorescence from more than 1 fluorochrome. (referred to as bleed over)Compensation needs to be made for this bleed over so that 1 detector reports signal from only 1 fluorochrome
50Setting Up Controls- Compensation FITC+ ControlParameter voltage must be altered to compensate for this overlapProper compensation used to be performed visually, but this has been found to be very wrongIn order to properly ensure compensation, must use Mean MatchingFITC Overlapping into the PE ChannelFITC Properly Compensated
51Setting Up Controls- Compensation In the Compensation panel, locate the channels requiring compensation (FL2-% of FL1, etc.)The positive control you are using is the first channel being subtracted from the second (FITC from PE as above)Place the positive control tube onto the bulk injection port check the Setup box and click AcquireBegin making small adjustments to the percentageAfter each adjustment, press Stop followed by Restart in either the Parameter Description or Acquisition panelWatch the Mean Fluorescence Intensity (MFI) of the positive control compared to the negativeContinue compensating until the mean of the positive population is nearly matched to the mean of the negativeOnce the means have been approximately matched, abort acquisition, uncheck the Setup box, and begin acquiring data
52BD CellQuest- Performing Your Experiment Once all controls are set and compensation has been performed (if necessary), start running unknown samplesBe sure that the Setup box is left uncheckedContinue acquiring until all samples have been runIf another appointment follows yours, do not shutdown the cytometerPlace a tube of water on the sample injection port and allow it to run for ~2 minutes.Press the Standby button on the front of the cytometerYou may now skip away in bliss at having successfully revolutionized your field of research with your staggeringly awesome data (DOUBLE HUZZAH)!!!
53FACSCalibur- Shut Down If you are the last appointment of the day, once all samples are run, place a tube of sterile water onto the sample injection portKeep the cytometer in Run modeLeave the tube on for ~2 minutesReplace the tube of water with the tube containing 10% bleach for ~2 minutesPlace the tube containing water back onto the cytometer for another 2 minutesPlace the cytometer in Standby modeTurn the computer offGo to File>ShutdownTurn the instrument off by pressing the illuminated green button on the back, right side of the cytometerSwitch off the fluidics cart by pressing the illuminated green button
55InterpretationOnce the values for each parameter are in a list mode file, specialized software can graphically represent it.The data can be displayed in 1, 2, or 3 dimensional formatCommon programs include…CellQuestBD FACSDivaFlowjoFCS ExpressBD Paint-a-GateEvery program has its Pros and Cons, most require licensingPaint-a-Gate is available on the workstation in the Flow Core
56Important Points on Analysis What kind of data are you looking for?How much fluorescence?What percent are positive?How much more positive is x than y?What is the ratio between param1 and param2What kind of statistics are availableMFI (geometric or arithmetic)%-agesCVMedianAnything you can do with a list of numbers
57Everything’s Relative The relative bin numbers are just that…relative.Saying your cells have a mean fluorescence intensity of 100 means absolutely nothing until you compare it to a negative.The fact that everything is relative allows you to compare 2, 3, or 20 samples using the same instrument settings.
58FACSCalibur- Troubleshooting Under normal weekday circumstances, the cytometer should be ready for useContact one of the operators to change the sheath or empty the wasteIf using the instrument on the weekend, set up will need to be performedIf the sheath is empty, Refill the sheath from one of the BD FACSFlow containers designatedIf the waste is full , empty the container into the sink and reconnect the cap/tubingIf any issues with the cytometer are encountered:DON’T PANIC!! (Always carry a towel)Remove any sample runningPlace the cytometer in StandbyContact one of the operatorsDo not try to fix the instrument under any circumstanceWe have a service contract for that
59AppointmentsFlow appointments should be scheduled through one of the Flow Core operatorsNo appointments byCall x4277Stop by HEB 270 or 211Please arrive on timeBilling begins at the time scheduledAdditional time for use will be allowed only if no other users are scheduled following the appointmentBilling is done in 15 minute incrementsCancellations should be made at least 24hrs in advanceCancellations without proper notice will be billed accordingly unless there are exigent circumstances
60References Numerous References available in the Flow Lab CytometryCurrent Protocols in Flow CytometryMany more reference books availablePurdue University Cytometry Laboratories website:Dr. Robert Murphy, Carnegie Mellon University- Basic Theory 1 and 2 PowerPoint slidesThe Scripps Research Institute Flow Cytometry Core Facility: