BD FACSAria™ III - BD Biosciences [PDF]

BD FACSAria III

Innovation is Built In

Innovation is built into the BD FACSAria III The BD FACSAria™ III is built on the solid foundation of patented technologies, superior multicolor performance, and legendary ease-of-use that has led to the unparalleled success of the BD FACSAria. Since the introduction of the first BD FACSAria in 2003, each successive generation has opened the complex world of cell sorting to a broader audience of researchers and wider range of applications. Now, the BD FACSAria III system is even more powerful, dependable, and easy to use. Here are a few of the new innovations that the BD FACSAria III has to offer: Superior Multicolor Performance To achieve superior multicolor performance, the fluidics and optical systems are precisely integrated to maximize signal detection. Innovations include the laser excitation optics, the patented flow cell with gel-coupled cuvette, and the highly efficient patented octagon and trigon detection system. These systems work in unison, allowing the BD FACSAria III to achieve unrivaled sensitivity and resolution. Expandable for the Future, Affordable Today The BD FACSAria III has flexibility built in. It can mount up to six lasers, so you can choose the configuration that meets your application investment and site requirements today— all the while knowing the system is expandable to up to six lasers to meet future needs. An innovative new X-mount optical plate accommodates easy expansion to six lasers and four spatially separated beam spots. Wavelength choices now include 561-nm and 445-nm lasers, as well as the 488-nm, 633-nm, 405-nm, and 375-nm lasers. Mount up to 20 detectors, and measure a maximum of 18 colors simultaneously. Smart, Long-Term Investment Unlike other instruments, the BD FACSAria enables customers to upgrade their existing instruments to the next generation platform instead of purchasing new systems. The field upgrade is another unique BD innovation that can bring your BD FACSAria or BD FACSAria II system up to the capabilities of the BD FACSAria III. This capability makes the BD FACSAria platform the best possible choice and a smart, long-term investment.

Innovation has always been built into the BD FACSAria. The new BD FACSAria III represents the latest innovations, offering advances that deliver reproducible results and superior performance. BD FACSAria III—in a perpetual state of the art to support your next great discovery.

FLUIDICS

Sensitivity for multicolor and sorting applications

Proven dependability and ease of use put the system in a class of its own The fluidics system in the BD FACSAria III cell sorter is pressure driven. Positive air pressure forces sample cells through an optically gel-coupled cuvette flow cell. Hydrodynamic focusing guides particles in a single-file stream through the cuvette, where laser light intercepts the stream at the sample interrogation point. Gel-coupled Cuvette Flow Cell At the heart of the BD FACSAria III is a quartz cuvette flow cell in true fixed alignment with the laser and gel coupled to the collection optics. For greater sensitivity, the BD FACSAria III incorporates a next-generation cuvette in the flow cell. Its patented design helps ensure that lasers are precisely focused on the sample stream, that they generate the greatest signal, and that the maximum amount of emitted light is collected. Fixed alignment minimizes startup time, improves experiment-to-experiment reproducibility, and enables automated daily quality control. Most importantly, it also improves collection efficiency and optimizes resolution needed for multicolor applications, even at high-speed sorting settings.

Four beam spots in the BD FACSAria III

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In addition to other benefits, the next generation flow cell in the BD FACSAria III is designed to improve resolution for side population applications and DNA cell cycle analyses. High-Performance Analysis, High-Performance Sorting The BD FACSAria III analysis performance is comparable to state-of-the-art highly sensitive analyzers. This is accomplished by using a similar gel-coupled flow cell design and the fixed optical architecture of the BD FACSCanto™ II and BD LSRFortessa™ systems. This design architecture achieves high numerical aperture light collection. The flow cell and nozzle design enable low particle speeds in the analysis zone for maximum light collection, and then accelerate the particle through the nozzle at stream speeds to achieve the drop rates required for high-performance sorting. Through the precise coordination of the optical and fluidics systems, the BD FACSAria III delivers exceptional optical detection sensitivity compared to traditional stream-in-air systems, in which particle speeds are the same for both analysis and sorting.

Nozzles for a Range of Particles A choice of nozzles lets users sort a wide range of particle sizes. Nozzles are available in four sizes: 70, 85, 100, and 130 microns. Nozzles are readily accessible and easy to change, with a design offering tight registration for a secure fit. This means a reproducible drop profile after every nozzle exchange, resulting in reproducible instrument setup and alignment. The software sort setup matches pressure and sort settings to the nozzle being used. Easy Aseptic Setup and Cleaning Innovations in the fluidics system such as easy-to-insert nozzles, automated sort setup, and easy-to-change filters make setup fast and simple. The fluidics design features integrated valve manifolds and a streamlined fluidics path. Software wizards make aseptic sort setup easy and effective. In addition, after a sample tube is run, both the inside and outside of the sample injection tubing are flushed to minimize carryover.

Cuvette flow cell and nozzle

BD Accudrop technology simplifies drop delay determination Patented BD FACS™ Accudrop technology assists the user to see the best drop delay value which is invisible to the naked eye. Software automation simplifies drop delay determination. Once the drop delay is calculated, the system automatically adjusts to maintain a constant break-off, called the Sweet Spot. Automatic clog detection stops the sort and protects the collection tubes if a clog is detected. After passing through the cuvette, the stream accelerates through the nozzle and droplets are formed for sorting. Since particle interrogation occurs above the nozzle, insertion and removal of the nozzle can occur without realigning the optics or the fluid stream.

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FLUIDICS

Engineered into the system

Fluidic system improvements are built in to make the system easier and safer to operate Fluidics Cart A self-contained fluidics cart supplies sheath and cleaning fluids and collects waste from the cytometer. The cart also provides the air pressure and vacuum needed to achieve pressure from 5 to 75 psi, to accommodate a variety of cell sorting applications. BD FACSDiva™ software adjusts the air pressure. The fluidics cart is typically positioned directly under or to the left of the cytometer. The fluidics cart holds a 10-L stainless steel sheath tank, a 5-L stainless steel tank used for shutting down the instrument with ethanol, and 10-L waste container. The sheath tank can be autoclaved. In addition, the cart holds three 5-L auxiliary cleaning fluid containers used in conjunction with the automated Prepare for Aseptic Sort mode.

Fluidics cart

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Gating strategy used to sort CD45RA+ and CD45RA- Tregs The BD FACSAria II system was set up for a sort using either a 70-μm or 100-μm nozzle (70 psi or 35 psi with a frequency + of 87 or 60 kHz respectively). Tregs and CD45RAB CD45RALymphocyte Gate All Events Tregs were sorted in purity mode at a rate of 10,000 to 11,000 events per second.

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Sample Injection Chamber During acquisition, the sample injection chamber is pressurized, forcing the sample to the cuvette flow cell. To simplify acquisition, the chamber temperature and agitation settings are controlled using BD FACSDiva software. A variety of tube holders are provided, from 15-mL centrifuge tube to 1.0-mL microtube size. To minimize clogging, 35and 50-micron sample line filters are available. Sort collection chamber

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30,000 22,506 22,394 22,302 11,137 944 422 515 2

#### 75.0 99.5 99.6 49.9 8.5 44.7 54.6 10

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HH Tube: Tube:Treg Tregcocktail cocktail

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All Events Lymphocyte Gate Doublet Disc 1 Doublet Disc 2 CD4 Gate CD127 lo Treg CD4+CD25+CD127lo CD45RA+ CD4+CD25+CD127lo CD45RA CD4+CD25+CD127lo CD45RA

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Aerosol Management Engineered with aerosol management in mind, the BD Doublet Disc 1 Doublet Disc 2 III features Doublet an enclosed pathway from the sample Lymphocyte FACSAria Gate Disc 1 Doublet Disc 2 Lymphocyte Gate SSC-A injection chamber to the sort collection tubes. For an FSC-A SSC-A FSC-A added level of aerosol management, the BD™ Aerosol F SSC-A Specimen_001-TregFSC-A cocktail FSC-A Management Option (AMO) evacuates the sort collection D E F Specimen_001-Treg cocktail CD4 Gate Doublet Disc 2 D E F Specimen_001-Treg cocktail chamber and traps aerosolized particles during sorting. CD4 Gate Doublet Disc 2 100

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The sort block also houses an aspirator drawer that keeps the sort collection tubes covered until sorting begins and automatically to protect the tubes when the Sweet Doubletcloses Disc 1 Doublet Disc 2 Spot is on and a clog is detected. 250 (x 1,000)

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Lymphocyte Gate The sort collection chamber’s universal mount design makes Doublet Disc 1 B Lymphocyte Gate inserting the tube holders easier. The holders areC designedDoublet Disc 1 to help maintain aseptic conditions when removing sort tubes. Temperature control for sort collection tubes, slides, and plates is available as an option.

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From the Sort Block to the Collection Chamber After the nozzle, particles pass C through the B leavingLymphocyte Gate Doublet Disc 1 sort block that houses the deflection plates. A The novel All Events design fixes the plates in position for moreAefficient and All Events reproducible deflection into a collection device in the sort collection chamber.

Population Population

100.0 75.0 74.6 74.3 37.1 3.1 1.4 1.7 4

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All AllEvents Events Lymphocyte Gate Lymphocyte Gate Doublet Disc 1 1 Doublet Disc Doublet Disc 2 2 Doublet Disc CD4 Gate CD4 Gate CD127 lo Treg CD127 lo Treg CD4+CD25+CD127lo CD45RA CD4+CD25+CD127lo CD45RA CD4+CD25+CD127lo CD45RA CD4+CD25+CD127lo CD45RA

#Events %Parent %Total #Events %Parent %Total 30,000 30,000 22,506 22,506 22,394 22,394 22,302 22,302 11,137 11,137 944 944 422 422 515 515

######## 100.0100.0 75.0 75.075.0 75.0 99.5 99.574.6 74.6 99.6 99.674.3 74.3 49.9 49.937.1 37.1 8.5 3.1 8.5 3.1 44.7 1.4 44.7 1.4 54.6 1.7 54.6 1.7

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OPTICS

Increased efficiency for multicolor detection

Sensitivity and resolution beyond compare Innovations in the optical system, pioneered by BD, efficiently maximize signal detection and greatly increase sensitivity and resolution for each color in a multicolor assay. Enhanced sensitivity and resolution mean that even dim populations can be readily identified and sorted. The optics system allows optimizing multicolor assays and panel design for superior results. The design allows choice of laser excitation wavelength(s) that illuminate cells in the sample, and collection optics that direct light scatter and fluorescence signals through spectral filters to detectors. Innovative designs for both the excitation and collection optics reduce excitation losses and dramatically improve collection efficiency, yielding better information from each sample.

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Excitation Optics The excitation optics consist of multiple fiber launched fixed-wavelength lasers, beam shaping optics, and achromatic focusing lenses that produce beam spots that are spatially separated and concentrated (9 μm x 65 μm). The more concentrated the beam spot, the higher the signal produced as each fluorescent labeled particle passes through the laser spot. Laser light is focused into the gel-coupled cuvette flow cell. Optical gel coupling to the fluorescence objective lens transmits the greatest amount of emitted light from the interrogation point to the collection optics. Since the optical pathway and the sample core stream are fixed, alignment is constant from day to day and from experiment to experiment. Fixed alignment also ensures that there is no variability in experiment results introduced by manual optical adjustments. Collection Optics Fiber optics deliver emitted light from the gel-coupled cuvette to the detector arrays. The collection optics are set up in patented octagon- and trigon-shaped pathways that maximize signal detection from each laser illuminated beam spot. This is accomplished by transmitting the highest wavelengths (which have the fewest photons of light) to the first photomultiplier tube (PMT), and reflecting lower wavelengths to the next PMT through a series of longpass dichroic mirrors.

A BD FACSAria III

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The 561-nm laser excites mCherry and PE more efficiently compared to the 488-nm laser. The same sample of stained mouse spleen cells was acquired using a BD FACSAria II (488-nm, 405-nm, and 633-nm lasers) and BD FACSAria III (488-nm, 405-nm, 633-nm, and 561-nm lasers). Dim staining populations can only be detected using the 561-nm laser.

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Trigon and octagon detector arrays

Precision Optical Design The many innovations in the BD FACSAria III’s optical system, such as the patented gel-coupled cuvette and octagon detection system, and the 9 μm x 65-μm beam spot, are designed to work together to maximize sensitivity and resolution. This precision design delivers a more efficient optical system enabling the use of lower powered lasers, which in turn reduces the total cost of instrument operation.

This design is based on the principle that light reflection is more efficient than light transmission. Emitted light travels to each PMT via reflection and is transmitted through only two pieces of glass to reach each detector. Therefore, colors can be detected with minimum light loss. Bandpass filters in front of each PMT allow spectral selection of the collected wavelengths. Importantly, this arrangement simplifies filter and mirror changes within the optical array and requires no further alignment for maximum signal strength.

CD45RA+ Tregs

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Representative FoxP3 staining of sorted Tregs To determine purity, as defined by FoxP3+ status, a portion of the cells was stained with anti-human FoxP3 BD Horizon™ V450. Data is representative of 10 experiments.

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OPTICS

Multicolor flexibility

The latest state-of-the-art is available with a simple upgrade Flexible for Present and Future Needs For many users, the capability of an advanced cell sorter is defined by its flexibility, which in turn is defined by the number of parameters that can be detected simultaneously. With flexibility built in, the BD FACSAria III can support six lasers and four spatially separated beam spots. Choose up to six laser wavelengths—633 nm, 561 nm, 488 nm, 445 nm, 405 nm, and 375 nm—and up to 20 detector positions, to measure up to 18 colors simultaneously.

Configure your BD FACSAria III Up to 6 lasers Up to 18 colors Laser choices: 633, 561, 488, 445, 405, 375 nm

Upgradeable and Backward Compatible Users can design a configuration that meets their lab’s budget and site requirements today and have a growth path for the future. They can expand the BD FACSAria III system with additional lasers via a field upgrade. In addition, customers with BD FACSAria or BD FACSAria II systems can upgrade them to the new capabilities of the BD FACSAria III with a field upgrade. Lower Cost of Ownership The unique, efficient design of the optical system delivers a lower cost of operation than stream-in-air sorters. The BD FACSAria III delivers maximum sensitivity and resolution using fiber launched solid state lasers. No special power or cooling is needed for these lower powered air cooled lasers.

CFP 445 Transfected-HeLa 100

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#### 56.9 90.1 97.8 31.7 68.3

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CFP-transfected HeLa cells The 445-nm laser excites Cyan Fluorescent Protein (CFP) more efficiently compared to the 405-nm laser. The same sample of CFPtransfected HeLa cells was acquired on the BD FACSAria III and excited either by the 445-nm laser or by the 405-nm laser. The cells excited by the 445-nm laser demonstrated improved separation compared to those excited by the 405-nm laser.

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#Events %Parent %Total 10,000 4,561 3,706 3,505 2,049 1,456

#### 45.6 81.3 94.6 58.5 41.5

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New X-mount Optical Plate

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Tube: mouse BM CD34KSL Hoechst 5 *0ug/ml Population

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All Events Cells PIDoublet Doublet-CD45+ Lineagec-Kit+ Sca-1 + LinSide population c-Kit+ Sca-1 + Lin- AND Side population

1,000,000 859,338 808,841 800,858 799,149 790,724 144,071 4,610 698 665

#### 100.0 85.9 85.9 94.1 80.9 99.0 80.1 99.8 79.9 98.9 79.1 18.2 14.4 3.2 0.5 0.5 0.1 0.5 0.1

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Cancer cell line side population Human HT-29 colon cancer cells were stained with Hoechst 33342 and acquired on the BD FACSAria III equipped with a 375-nm laser (left dot plot). As a control, side population expression was blocked (right dot plot).

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#Events %Parent %Total 20,000 19,525 19,264 18,997 18,427 650 7,395 2,904

#### 97.6 98.7 98.6 97.0 3.5 40.1 15.8

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Antibodies used were c-Kit PE, Sca-1 PE-Cy™7, Lineage FITC, CD34 APC, CD45 APC-Cy7, PI, Hoechst Blue, and Hoechst Red.

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The SP fraction (red) was limited to those cells expressing c-Kit+ Sca-1++.

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Mouse bone marrow side population Mouse bone marrow cells were stained with Hoechst 33342, c-Kit, Sca-1, and lineage markers, and run on the BD FACSAria III equipped with a 375-nm laser.

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CONTROLS AND

Compatible with other BD analyzers and sorters

BD FACSDiva software helps you move from analysis to sorting BD FACSDiva software efficiently controls the setup, acquisition, and analysis of flow cytometry data from the BD FACSAria III workstation. BD FACSDiva software is common across many BD cell analyzers and cell sorters, including BD FACSCanto and BD™ LSR systems. Researchers gain application flexibility because it is easier to move the assay design and optimization to another platform, for example, from analysis to sorting.

Aria II CD4 FITC

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(x 1,000)

250

50

FACSCanto II

Aria II CD4 Pacific Blue

250

(x 1,000)

FACSAria II

Aria II CD4 APC

250

SSC-A

Platform comparison using CD4 Comparison of whole blood stained with single-color CD4 FITC, CD4 APC, and CD4 Pacific Blue™ and run on both the BD FACSAria II and BD FACSCanto II systems. The BD FACSAria II was set for high-speed sorting (70 psi and 90 kHz), and both instruments were set up using BD Cytometer Setup and Tracking software.

(x 1,000)

y Perform the sort.

SSC-A

x Optimize the sample.

(x 1,000)

wP  erform automated setup, QC, and drop delay optimization.

Acquisition and Analysis BD FACSDiva software enables researchers to preview and record data from multiple samples with an automated acquisition process. The software manages acquisition templates, experiment layouts, and compensation procedures to further facilitate data acquisition.

SSC-A

v Start the fluidics.

(x 1,000)

u Turn on the sorter.

SSC-A

Fast startup to sort time

The Cytometer Setup and Tracking (CS&T) feature of BD FACSDiva software establishes baseline settings and optimizes instrument sensitivity and fluorescent resolution. The software reduces the chances of operator error, and ensures consistency of results. It allows for the creation of application-specific settings for rapid performance of routine experiments in a more consistent manner. Tracking capabilities in the software measure a number of instrument settings and report on performance, simplifying daily quality control. Levey-Jennings plots help users understand instrument performance and identify maintenance issues.

102

103

APC-A

104

105

0 -39

102

103

104

Pacific Blue-A

105

A N A LY S I S

For efficient and convenient analysis, the software provides automated hierarchical snap-to gating, user selectable plot configurations, and batch analysis function. Recorded data can be analyzed by creating plots, gates, population hierarchies, and statistical views on a BD FACSDiva global worksheet. Once the global worksheet is saved, it can be used to analyze multiple sample tubes from an experiment, thereby saving time. Other productivity benefits come from features such as user-definable batch analysis and automated gate resizing, pausing between data files, exporting statistics, and printing before proceeding to the next data file. Digital Electronics The gel-coupled cuvette and electronics operate together to deliver the maximum amount of signal information about each particle. The electronic sampling rate is precisely matched to the speed of the particles flowing through the cuvette. The BD FACSAria III electronic design has demonstrated linear and accurate event data acquisition at up to 70,000 events per second (shown in graph below). BD Cytometer Setup and Tracking software

Proccessed Events

Outperforms analog from 0 to 100,000 events/sec

90000 80000 70000 60000 50000 40000 30000 20000 10000 0

BD FACSAria III: uncompensated BD FACSAria III: compensated Dead time of 5.5 �sec BD FACSDiVa: compensated

0

30000

60000

90000 120000 150000 180000 210000

Event Rate

BD FACSAria III system acquisition rates

13

SERVICES

Services Field Service When instrument installation or service is required, a BD Biosciences Technical Field Service Engineer can be dispatched to the customer site. BD Biosciences field service engineers are located across the world. On-site service and maintenance agreements are available to provide long-term support for the BD FACSAria III.

BD Biosciences is fully committed to the success and satisfaction of its customers. The BD FACSAria III cell sorter is backed by a world-class service and support organization with unmatched flow cytometry experience. Since commercializing the first cell sorter in 1973, BD has been delivering flow cytometry systems that are even more powerful, dependable, and easy to use. This expertise is made available to BD FACSAria III customers through comprehensive training, applications and technical support, and expert field service.

Custom Services Mobilizing technology for research applications requires close collaboration. The Custom Technology Team (CTT) at BD Biosciences works with customers to provide solutions through custom reagents, panels, or assay protocols.

Training Hands-on training is included with each BD FACSAria III cell sorter. In addition, training courses are held at BD training centers worldwide. BD flow cytometry courses combine theory and practice to provide participants with the skills and experience they need to take full advantage of the capabilities of the BD FACSAria III cell sorter.

Staffed by leading scientists with breadth and depth of technical expertise in cytometry, the CTT team will coordinate with researchers to study the problem at hand, make recommendations, and help implement the solutions. In this way, BD Biosciences technical know-how is translated into practical solutions that allow customers to focus on research.

Technical Application Support BD Biosciences technical applications support specialists are available to provide field- or phone-based assistance and advice. Expert in a diverse array of topics, BD technical application specialists are well equipped to address customer needs in both instrument and applications support.

Special Order Research Products Instruments can be customized to meet customer requirements via the Special Order Research Products Tube: Differentiating H9 #Events %Parent %Total (SORP) programs. Population Tube: Differentiating H9

10

5

Differentiating H9

10

4

5

Differentiating H9

0

SSEA-1 FITC-A SSEA-1 FITC-A SSEA-1 FITC-A SSEA-1 FITC-A 2 3 4 10

10

10

SSEA-1 FITC-A

Population Population Population Population Gated Gated cellscells Gated cells Gated cells SSEA-3+TRA-1-81+ SSEA-3+TRA-1-81+ Population SSEA-3+TRA-1-81+ SSEA-3+TRA-1-81+ SSEA3-TRA1-81SSEA3-TRA1-81SSEA3-TRA1-81SSEA3-TRA1-81Gated cells P1 P1 P1 P1 P2 P2SSEA-3+TRA-1-81+ P2 P2 P3 P3SSEA3-TRA1-81P3 P3

10

5

5 5 105 10 5 10

10 10 647-A 10 TRA-1-81 Alexa TRA-1-81 Alexa 647-A 2 Alexa 3 647-A 4 TRA-1-81 Alexa TRA-1-81 0 10 10647-A 10

P2 4

3

5

-226

#Events #Events #Events #Events 8,490 8,490 8,490 8,490 3,433 3,433 #Events 3,433 3,433 1,876 1,876 1,876 1,876 8,490 2,015 2,015 2,015 2,015 3,433 6,965 6,965 6,965 6,965 7,283 1,876 7,283 7,283 7,283

2,015 6,965 7,283

0 10

10

10

4

10

5

SSEA-3 PE-A

%Parent %Parent %Parent %Parent 84.984.9 84.9 84.9 40.440.4 40.4 %Parent 40.4 22.122.1 22.1 22.1 23.723.784.9 23.7 23.7 82.082.040.4 82.0 82.0 85.885.822.1 85.8 85.8

23.7 82.0 85.8

Count

Count Count Count

2

SSEA-3 PE-A SSEA-3 2 PE-A3

10 10

4

5

10

3

10

4

10

34.3 100.0 #### 18.8 84.9 84.9 20.2 34.3 40.4 69.6 18.8 22.1 72.8 20.2 23.7 82.0 69.6 85.8 72.8

5

10

2

10

4

10

10

SSEA-1 FITC-A 0

2

10

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3

P3

-226

4

10

10

2

0

5

10

0 10

3

0 10

10

10

4

P2 10

5

SSEA-3 PE-A

-226

0 10

2

#Events 8,490 3,433#Events 1,876 8,490 2,015 3,433 6,965 1,876 7,283 2,015 6,965 7,283

10

4

10

10

3

10

10

5

4

10

5

TRA-1-81 Alexa 647-A Differentiating H9

Differentiating H9 P2

2

3

2

10

Count

Count

10

SSEA-1 FITC-A

Population 5 SSEA-3+TRA-1-81+

4

P1 5

10

TRA-1-81 Alexa 647-A

3

10

0 1020304050607080

4

2

-100

4

5

10

SSEA-3 PE-A %Parent 84.9 %Parent 40.4 22.1 84.9 23.7 40.4 82.0 22.1 85.8 23.7 82.0 85.8

Differentiating H9 0 1020304050607080

10

3

0 1020304050607080

2

10

-100

Differentiating H9

Count

Differentiating H9 P1

0

-100

5

10

Count

10

5

-226

10

-226

10

4

10

4 3

SSEA-3 PE-A

10

3

10

Gated cells

3

10

SSEA-3 PE-A

2

0 10

0

3

0 1020304050607080

4

10

Population 10

5

5

10

4

10

3

10

3

2

SSEA3-TRA1-81Gated cells TRA-1-81 Alexa 647-A Differentiating Differentiating H9 H9 Differentiating Differentiating H9 H9 P1 SSEA-3+TRA-1-81+ P2 SSEA3-TRA1-81Differentiating H9 Differentiating H9 P3 P1 P3 P2 P2 P3 P2 P2 P3 P3 P2 P3

2 3 4 5 0 1020 10 2 103 103 104 104 105 105 -226-226 10 0 100 10 10 10 10 10 -226-226 SSEA-3 PE-A 10 SSEA-3 PE-A

10

2

40.4 10,000 22.1 8,490 23.7 3,433 82.0 1,876 85.8 2,015 6,965 7,283

SSEA-3 PE-A

2

10 0

-42 50 100 150

0

0

10 -100

0 1020304050607080

0

-42

-100-100

0 1020304050607080 0 1020304050607080

P1 P1 P1 P1

5

Differentiating Differentiating H9 H9 Differentiating Differentiating H9 H9

1020304050607080 00 1020304050607080 Count

Count Count Count

0 1020304050607080 0 1020304050607080 Count

0 0 50Count 50100 100150 150 Count Count

50150 100 150 500 100 0 50 100 150

Differentiating H9

P1 2 2 3 103 4 104 5 105 0 010 10 2 10 3 10 4 10 5 -42 0-42010210 10310 10410 10510 -42 -42

0 10

10 0 1020304050607080 Count 0 1020304050607080

2 3 4 TRA-1-81 Alexa 647-A Alexa 0 TRA-1-81 10 10 647-A10

TRA-1-81 Alexa 647-A Differentiating Differentiating H9 H9 Differentiating Differentiating H9 H9

0 1020304050607080

-100

4 4 104 10 4 10

10

TRA-1-81 Alexa 647-A

-42

3 3 103 10 3 10

10

SSEA-3 PE-A

0 10 10 -226 Differentiating H9

3,433 1,876 2,015 6,965 7,283

5

5

10

-42

Count

50 100 150 0

10

Count

10

5 3

-42

2 2 0 0102 10 2 -100-100 0 010 10

3

0 10

-226 250 (x 1,000)

Alexa 647-A DifferentiatingTRA-1-81 H9

2

5 5 105 10 5 10

10

-100

4

SSEA-3 SSEA-3PE-A PE-A

-42 -42 0

4 4 104 10 4 10

2

0

-100

Differentiating H9

-226 -226 2 2SSEA-3 3 4 0 10 10103 PE-A 10104 SSEA-3 PE-A 0 10 -226 22 33 4 -226 0 10SSEA-3 10 PE-A 10 4 0 10 10 10 -226

2

3

4

SSEA-1 SSEA-1FITC-A FITC-A

2 10FITC-A 3FITC-A SSEA-1 SSEA-1 10 10 4

5

10105 5 10 5 10

5 10 5

SSEA-1 10 FITC-A 10 10 -42 -42-42 0 2 10 2 3 3 44 55 10 210 10 3 10 10 4 1010 5 0 010 0 10 10 10 10

3 3 103 10 3 10

10 10 647-A 10 TRA-1-81 Alexa TRA-1-81 Alexa 647-A

-100-100

Count Count

5

10

Differentiating Differentiating H9 H9 Differentiating Differentiating H9 H9

Differentiating H9

2 2 0 0102 10 2 -100-100 0 010 10

14

10

3

0

Differentiating Differentiating H9 H9 Differentiating Differentiating H9 H9

P1 P2 P3

10

2

10

2

10 0

SSEA-3 PE-A PE-A SSEA-3 PE-A SSEA-3

SSEA-1 FITC-A

SSEA-1 FITC-A

4

5

10 10 10

3

SSEA-1 SSEA-1FITC-A FITC-A

4

-42 -42 22 3 FITC-A 4 0 1010SSEA-1 10103 10104 SSEA-1 FITC-A -42 0 22 3 4 -42 0 10 SSEA-1 10 3FITC-A 10 4 0 10 10 10

-42

2 2 3 3 4 4 5 5 100 100 150 150 200 200 250 250 0 1002 10 103 10 10 2 3 310410104 45 105 250250 -226-2260 10 100100 150150 250 (x 1,000) 0 100 10210 10 1,000) 10 100FSC-A 150200200(x 200 10PE-A 1010 10 SSEA-3 FSC-A -226-226-226 SSEA-3 PE-A (x 1,000) (x 1,000) FSC-A (x 1,000)

FSC-A FSC-A

200

10,000 #### 100.0 %Parent 8,490#Events 84.9 84.9%Total

Differentiating H9 Differentiating H9 Tube: Differentiating Tube: Differentiating H9 Tube: Differentiating H9 H9 Tube: Differentiating Tube: Differentiating H9H9 Population #Events %Parent %Total %Total %Total Population #Events %Parent #Events %Parent Population %Total Population #Events %Parent %Total Population #Events %Parent Events 10,000 #### ####100.0 100.0 All All Events 10,000 All Events 10,000 #### 100.0 100.0 Events 10,000 #### AllAll Events 10,000 Gated cells cells 8,490 #### 84.9100.0 Gated cells 8,490 84.9 84.984.9 Gated 8,490 84.9 84.9 Gated cells 8,490 84.9 84.9 84.9 Gated cells 8,490 SSEA-3+TRA-1-81+ 3,433 40.440.4 84.9 SSEA-3+TRA-1-81+ 3,433 34.334.3 SSEA-3+TRA-1-81+ 3,433 3,433 40.4 34.3 SSEA-3+TRA-1-81+ 3,433 40.4 34.3 SSEA-3+TRA-1-81+ SSEA3-TRA1-811,876 40.4 22.1 34.3 SSEA3-TRA1-811,876 22.1 18.818.8 18.8 SSEA3-TRA1-811,876 22.1 SSEA3-TRA1-811,876 22.1 22.1 18.8 18.8 SSEA3-TRA1-811,876 2,015 23.723.7 20.220.2 P1 P1 2,015 P1 2,015 23.7 20.2 2,015 23.7 20.2 20.2 P1 P1 2,015 6,965 23.7 P2 P2 6,965 82.082.0 69.669.6 6,965 82.0 82.0 69.6 69.6 P2 6,965 82.0 69.6 P2 P2 6,965 7,283 85.8 85.8 72.872.8 P3 P3 7,283 7,283 85.8 85.8 72.8 72.8 P3 P3 7,283 72.8 P3 7,283 85.8

5

10105 5 10 5 10

(x(x 1,000) SSC-A 1,000) SSC-A 5050 100 150 250 (x(x1,000) 1,000) 100 150 200 200 250 SSC-A SSC-A (x 1,000) SSC-A 150 200 250 5050 100 100 250 100 150 150 200 200 250

50 50 50 50 50

2

250 (x 1,000)

150

0

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FSC-A

Differentiating Differentiating Differentiating H9 H9 H9 Differentiating Differentiating H9 H9

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FSC-A Differentiating H9

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Differentiating H9 Differentiating Differentiating H9 H9 Differentiating Differentiating H9 H9

2

-42

50

0

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2

3

150

100

SSC-A

50

50

All Events Population Gated cells SSEA-3+TRA-1-81+ All Events SSEA3-TRA1-81Gated cells P1 SSEA-3+TRA-1-81+ P2 SSEA3-TRA1-81P3 P1 P2 P3

4

10

SSEA-1 FITC-A

SSC-A

150

(x 1,000) 200 250

Differentiating H9

SSEA-1 FITC-A

(x 1,000) 200 250

Differentiating H9

0 10

P3

2

3

10

10

4

P3 5 10

TRA-1-81 Alexa 647-A -100

0 10

2

10

3

10

4

5

10

Alexa 647-A SSEA-3 TRA-1-81 PE-A TRA-1-81 Alexa 647-A SSEA-1 FITC-A Mean Mean Mean SSEA-3 PE-A TRA-1-81 Alexa 647-A SSEA-1 6,473 2,733 FITC-A 19,095 Mean Mean 10,223 273 34,080 Mean 167 6,473 9,462 2,733 271 19,095 2,852 10,223 10,977 14,141 34,080 273 7,872 1,484 9,462 22,258 167 271 7,390 2,852 2,495 10,977 22,242 14,141 7,872 1,484 22,258 7,390 2,495 22,242

5

2 3 4 5 0 1002 10 2 103 103 104 104 105 105 -100-100 0 10 10 10 10 10 10 10 0 10 TRA-1-81 Alexa 647-A -100-100TRA-1-81 Alexa 647-A

TRA-1-81 Alexa 647-A TRA-1-81 Alexa 647-A 2 3 4 5 0 10 10 10 10 -100 SSEA-3 PE-A TRA-1-81 Alexa 647-A SSEA-1 FITC-A TRA-1-81 SSEA-3 PE-A Alexa 647-A SSEA-1 FITC-A TRA-1-81 AlexaSSEA-1 647-A SSEA-3 PE-A TRA-1-81 Alexa 647-A SSEA-1 FITC-A SSEA-3 PE-A Alexa 647-A FITC-A Mean Mean TRA-1-81 Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean 6,473 2,733 19,095 6,473 2,733 19,095 SSEA-3 PE-A TRA-1-81 Alexa 647-A SSEA-1 6,473 2,733 FITC-A 19,095 6,473 2,733 19,095 10,223 273 34,080 10,223 273 34,080 Mean 10,223 Mean 273 Mean 34,080 10,223 273 34,080 9,462 167167 9,462 271271 167 6,473 9,462 2,733 271 19,095 167 9,462 271 2,852 10,977 14,141 2,852 10,977 14,141 2,85210,223 10,977 14,141 34,080 2,852 10,977 14,141 273 7,872 1,484 22,258 7,872 1,484 22,258 7,872 1,484 22,258 7,872 1,484 22,258 7,390 167 2,495 9,462 22,242 271 7,390 2,495 22,242 7,390 2,495 22,242 7,390 2,495 22,242

Differentiating H9 cells stained and run on the BD FACSAria II using the BD Stemflow™ Human Pluripotent Stem Cell Sorting and Analysis Kit. This kit contains three different fluorescent antibodies that can be used to identify both undifferentiated (TRA-1-81 and SSEA-3) and differentiated (SSEA-1) pluripotent stem 2,852 10,977 14,141 7,872 1,484 22,258 cells. This combination of markers has been widely used to characterize and isolate 7,390 2,495 22,242 differentiated and undifferentiated stem cells derived from hESCs and iPS cells.

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