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BD LSRFortessa Performance without peer,choice without compromise

Performance without peer,choice without compromiseThe BD LSRFortessa cell analyzer offers the ultimate in choice for flowcytometry, providing power, performance, and consistency. Designed tobe affordable and expandable, the BD LSRFortessa has the flexibility tosupport the expanding needs of multicolor flow cytometry assays.The BD LSRFortessa analyzer can be configured with up to 4lasers—blue, red, violet, and UV—which enables the detection of upto 18 colors simultaneously. The BD LSRFortessa may be upgradedsubsequently with additional or new lasers from BD, as futurerequirements dictate.Innovative Designs for Optical EfficiencyBuilt on a proven platform, BD LSR analyzersfeature innovative designs for both theexcitation optics and collection optics thatreduce excitation losses and improve lightcollection efficiency. The result is opticalefficiency that delivers maximum sensitivityand resolution for multicolor applicationsby reducing excitation laser light loss, andimproved fluorescent signal collection.Special Order ProgramFor research needs at the leading edgeof biomedical discovery, the special orderprogram offers BD LSRFortessa researchersthe latest innovations in laser technologyto enable future upgrades with newexcitation, wavelength, and power choices.The program lets customers fully customizeconfigurations to deliver added flexibilityand power to support advanced research.Technical and Application SupportAs with all BD instruments and reagents,highly qualified BD technical and applicationsupport personnel are available for helpin streamlining research and maintainingoptimal instrument performance.

Boundless PotentialThe BD LSRFortessa AnalyzerFlexibility in a Small SpaceAn affordable choice to fit most flow cytometry analyzerneeds, the BD LSRFortessa can be ordered with up to 4lasers—blue, red, violet, and UV—which provides flexibilityin laser wavelengths so assay design can be optimized usingthe latest fluorescent dyes and substrates. The instrumentcan accommodate the detection of up to 18 colorssimultaneously with a defined set of optical filters thatmeet or exceed the majority of today’s assay requirements.The BD LSRFortessa is designed to enable your current assayrequirements. As future needs arise, lasers can be added orupgraded.The BD LSRFortessa puts the power of the proven BD LSRplatform into a compact footprint. It can easily fit on thebenchtop for cost-effective space use. This space efficiencyis even more important for labs with limited space or wherespace cost is premium.In addition to the reduced size, design enhancementsprovide easier access to bandpass filters and mirrors,simplifying changes to experimental setup.To improve experimental workflow, the optionalBD High Throughput Sampler (HTS) provides rapid, fullyautomated sample acquisition from microtiter plates. Inhigh-throughput mode, the HTS option can speed througha 96-well plate in less than 15 minutes with less than 0.5%sample carryover from one well to the next. Low carryoveris essential in research applications to ensure sample purityand data integrity.BD High Throughput Sampler (HTS)Analysis of Regulatory T Cells4All EventsLymphocytes1053,500 4,000CD3 1021031041051041031,00050000CD8 PerCP-Cy5.52,500CD3 1,500 2,000Count3,000(x 1,000)250200CD4 CD8neg102CD3 APC-H70Lymphocytes50100150FSC200250(x 1,000)-132SSC150100-14050Regulatory T cells (Tregs) play a critical rolein maintaining immune modulation and arepresent in normal peripheral blood in lownumbers (5 to 10% of CD4 T cells or 1 to2% of total lymphocytes). Forkhead box P3(FoxP3) is considered to be the definitivemarker for this rare cell population.Tregs are known to express the highestlevels of CD25. The CD4 CD25 (high) gatingstrategy shown here was used to identifyTreg populations. These plots representdata taken from a BD LSRFortessa usingan 8-color panel that applies this gatingstrategy to identify FoxP3-positive cells.0 102102103104CD4 BD Horizon V500105

BD LSRFORTESSACD25 CD127loCD4 CD8negCD25 CD127loCD4 CD8neg-471-102 0 102103CD25 APC104105104103IFNγ CD4 #Events %Parent 69917.20.11,9213.00.2102-9210100102103104FoxP3 Alexa Fluor 4881050-4710-194-102-102010102CD25 CD127loPopulationAll EventsLymphocytesCD3 CD4 CD8negCD25 CD127loFoxP3 CD45RA CD25 CD45RAnegCD25 IFNg CD4 102CD4 BD Horizon V500103CD25 APC402030Count104103CD127 PECD45RA CD25 10450CD45RAnegCD25 FoxP3 Stimulated10510560105Tube:-313-102 0 102103104CD45RA BD Horizon V450105-265-102 0 102103104105IFNγ PE-Cy75

FLUIDICSSuperior PerformanceUnique and Revolutionary Designsfor Multicolor AnalysisFluidics SystemThe fluidics system is pressure driven. Hydrodynamic focusingforces sample cells through the cuvette flow cell, wherethey are interrogated. The flow cell is in fixed alignmentwith the laser and gel-coupled to the collection optics.This design ensures that the laser is precisely focused onthe sample stream and the maximum amount of emittedlight can be collected for added sensitivity in multicolorapplications. Fixed alignment also minimizes startup time,improves experiment-to-experiment reproducibility, andenables automated daily quality control.Many innovations are incorporated into the BD LSRFortessaproduct line. The heart of the cytometer, the fluidics systemfeatures a true fixed-alignment flow cell that is gel-coupledto the collection optics to maximize detector signal.The sheath container (8 L) and waste container (10 L) areoutside the cytometer, and positioned on the floor foreasier access.The optional BD FACSFlow supply system fluidics cartincreases capacity and ease of use while maintaining a stablefluidics pressure. It includes an automated sheath and wastefluid control system that reduces daily maintenance byincorporating two 20-L containers (Cubitainers ).Fluidic sensors maintain constant pressure, while a fluidicsmonitoring system warns when sheath fluid is low or empty,or when the waste container is full.Figure A-1 Hydrodynamic focusing of the sample core through the flow cellLow SamplePressure(12 µL/min)Laser BeamHigh SamplePressure(60 µL/min)Laser BeamFigure A-1 Hydrodynamic focusing of the sample core through the flow cellLow SamplePressure(12 µL/min)Laser SheathFluidHigh SamplePressure(60 µL/min)Laser heathFluidHydrodynamic focusing of the samplecore through the flow cell

Cuvette flow cellCuvetteLaser BeamsCuvette flow cellFlow CellA quartz cuvette flow cell is gel-coupledto the collection optics. This design helpsensure that lasers are precisely focused onthe sample stream to generate the greatestsignal and ensure that the maximumamount of emitted light is collected.Sample Injection Tube (SIT)7

OPTICSMaximum Signal, Minimum CrosstalkAn Innovative and Proven Platformfor Multicolor AnalysisOptics SystemThe optics system consists of laser excitation optics thatilluminate cells in the sample, and collection optics thatdirect light scatter and fluorescence signals through spectralfilters to detectors. Innovative designs for both the excitationoptics and collection optics in BD LSRFortessa systems reduceexcitation losses and optimize collection efficiency forincreased sensitivity and resolution.The patented collection optics are yet another designinnovation. Arranged in octagon- and trigon-shaped opticalpathways, their novel design efficiently maximizes signaldetection and increases sensitivity and resolution. Thisallows researchers to identify cells, especially dim and rarecell populations, optimizing multicolor assays and paneldesign for superior results.Excitation OpticsThe excitation optics consist of multiple fixed wavelengthlasers, beam shaping optics, and individual pinholes whichresult in spatially separated beam spots.A final lens focuses the laser light into the gel-coupledcuvette flow cell. Since the optical pathway and the samplecore stream are fixed, alignment is constant from day to dayand from experiment to experiment.Normalized Emission (%)Emission spectra of commonly used fluorochromesPerCP100PEAmCyan80Alexa Fluor 700BD Horizon V450FITCAPC60APC-H7PerCP Cy 5.540PE-Cy 72004008500600700Wavelength (nm)800

Collection OpticsEmitted light from the gel-coupled cuvette is deliveredby fiber optics to the detector arrays. The collection opticsare set up in patented octagon- and trigon-shaped opticalpathways that maximize signal detection resulting fromeach laser illuminated beam spot. Bandpass filters in frontof each PMT allow spectral selection of the collectedwavelengths. Importantly, this arrangement allows filterand mirror changes within the optical array to be madeeasily and requires no additional alignment for maximumsignal strength.This design is based on the principle that light reflection ismore efficient than light transmission. Emitted light travels toeach PMT detector via reflection and is transmitted throughonly two pieces of glass to reach the detector. Therefore,more colors can be detected with minimum light loss.DBFTransmission pathways in an octagonHGAEC9

Greater ConsistencyAutomated Controls for Setup,Acquisition, and AnalysisCytometer Setup and TrackingThe Cytometer Setup and Tracking (CS&T) feature ofBD FACSDiva software establishes baseline settings andadjusts for instrument variability. The software reducesoperator error, and ensures consistency of results by settingthe signal time delay across the multiple laser beams andoptimizing PMT voltages. Application-specific settings canalso be set, allowing for rapid setup and performanceof routine experiments in a more consistent manner.Quality control (QC) tracking capabilities in the softwaremeasure instrument settings and report on performance.Levey-Jennings plots help users understand instrumentperformance and identify maintenance issues.BD FACSDiva software controls the efficient setup,acquisition, and analysis of flow cytometry data fromthe BD LSRFortessa workstation. The software is commonacross BD FACS instrument platforms, including theBD FACSCanto cell analyzer and BD FACSAria cellsorter systems. This affords researchers greater applicationflexibility, allowing them to easily move assays from oneplatform to another.Analysis of human bloodData shows a 10-color panel run on a 3-laser BD LSRFortessa witha blue/red/violet configuration. The panel was used for definingT-cell subsets using lysed whole blood (BD Pharm Lyse lysingbuffer) from a normal donor. Use of the BD Horizon V450 andBD Horizon V500 dyes allows brighter fluorochromes to be used formore dimly expressed antigens, while the use of 10 colors expandsthe amount of information gained from a single tube.101050 102103TCR αβ FITC104105105104103CD8 PerCP-Cy5.510400105CD8 T Lymphs103CD8 PerCP-Cy5.5104103102-466T Lymphs0103104CD3 Alexa Fluor 700105-4660103104CD3 Alexa Fluor 700105-1080 102103104CD4 BD Horizon V500105-45104T Lymphs-693103CD45 APC-H7CD4 T Lymphs-693102T Lymphs0150SSC10050101-108CD4 BD Horizon V500105250T Lymphs200(x1,000)T Lymphs-1080103104CD3 Alexa Fluor 700105