流式细胞仪使用说明书

BD_FACSAria_III_brochure[1]

BD FACSAria III

Innovation is Built In

BD_FACSAria_III_brochure[1]

BD_FACSAria_III_brochure[1]

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:

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.

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.

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.

BD_FACSAria_III_brochure[1]

FLUIDICS

Sensitivity for multicolor and sorting applications

Proven dependability and ease of use put the system in a class of its ownThe 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 quart

z 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. 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.

Four beam spots in the BD FACSAria III

BD_FACSAria_III_brochure[1]

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 d

rop 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.

BD_FACSAria_III_brochure[1]

FLUIDICS

Engineered into the system

Fluidic system improvements are built in to make the system easier and safer to operateFluidics 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). CD45RA+ Tregs and CD45RAB Lympho

cyte 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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BD_FACSAria_III_brochure[1]

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. From the Sort Block to the Collection Chamber After leaving the nozzle, particles pass C through the B Lymphocyte Gate Doublet Disc 1 sort block that houses the deflection plates. The novel A All Events design fixes the plates in position for moreA efficient and All Events reproducible deflection into a collection device in the sort collection chamber.(x 1,000) 250 (x 1,000) 250(x 1,000) 200 1,000) 250 (x 250

Sort collection chamber

The sort block also houses an aspirator drawer that keeps the sort collection tubes covered until sorting begins and automatically closes to protect the tubes when 2 the Sweet Doublet Disc 1 Doublet Disc Spot is on and a clog is detected.150

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Aerosol Management Engineered with aerosol management in mind, the BD Doublet Disc 1 Doublet Disc 2 an enclosed pathway from the sample Lymphocyte FACSAria III features Doublet Disc 1 Gate 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 F chamber and E traps aerosolized particles during sorting. Specimen_001-Treg cocktail CD4 Gate Doublet Disc 2100

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mount design makes Doublet Disc 1 The sort collection chamber’s Gate B C Lymphocyte Gate Doublet inserting the tube holders easier. The holders are designed 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.(x 1,000) 250 (x 1,000) 250 (x 1,000) 250

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H HTube: Treg cocktail Tube: Treg cocktail Population Population All Events All 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 CD45RA105

PopulationCD4+CD25+CD127lo CD45RA-

#Events%Parent%Total 30,000 22,506 22,394 22,302 11,137 944 422 5152

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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 CD45RACD25 PE-A 3 CD25 PE-A 10103

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#### 75.0 99.5 99.6 49.9 8.5 44.7 54.6103

100.0 75.0 74.6 74.3 37.1 3.1 1.4 1.7104

#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######## 100.0 100.0 75.0 75.0 75.0 75.0 99.5 99.5 74.6 74.6 99.6 99.6 74.3 74.3 49.9 49.9 37.1 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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BD_FACSAria_III_brochure[1]

OPTICS

Increased efficiency for multicolor detection

Sensitivity and resolution beyond compareInnovations 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. 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 (9μm x 65μm). 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.

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Transmission pathways in an octagon

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A E CBD FACSAria II 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. 488-nm (blue) laserSpecimen_001-Sample 1150

BD FACSAria III 561-nm (yellow-green) laserSpecimen_001-Tube 001100

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BD_FACSAria_III_brochure[1]

Trigon and octagon detector arrays

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 tw

o 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.

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 9μm 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.CD45RA+ Tregs105 0 104 PE CD25 100 95.8 Relative Cell Number 102 103 104 FITC CD45RA 105 80 60 40 20 0 0 0 102

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CD45RA+ Tregs+CD45RA Tregs

CD45RA- Tregs CD45RA Tregs83.7 Relative Cell Number 80 60 40 20 0 PE CD25 104 100 105

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.

105 0 104 PE CD25

100 6.73 Relative Cell Number 102 103 104 FITC CD45RA 105 80 60 40 20 0 0 102 102 104 V450 FoxP3 105

95.8

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CD45RA- Tregs105 83.7 104 PE CD25 100 6.73 Relative Cell Number 80 60 40 20 0 0 102 102 104 105

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BD_FACSAria_III_brochure[1]

Protein (CFP) more efficiently compared to

the 405-nm laser. The same sample of CFP-

-1,144445 CFP-A

BD_FACSAria_III_brochure[1]

Hoechst Blue-A(x 1,000)250

100150200Cancer 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

500Hoechst Blue-A(x 1,000)002050HT-29 Hoechst 5ug/ml-PI-HT-29 Hoechst 5ug/ml Res 20ug/ml-PI-50100150200250(x 1,000)Hoechst Red-A50100150200250(x 1,000)Hoechst Red-A

BD_FACSAria_III_brochure[1]

CONTROLS AND

Compatible with other BD analyzers and sorters

BD FACSDiva software helps you move from analysis to sortingBD 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. 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. 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.

Fast startup to sort timeu Turn on the sorter. v Start the fluidics. w Perform automated setup, QC, and drop delay optimization. x Optimize the sample. y Perform the sort.

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.

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BD_FACSAria_III_brochure[1]

A N A LY S I S

For efficient and convenient analysis, the software provides automated hierarchical snap-to gating, us

er 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

Outperforms analog from 0 to 100,000 events/sec

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BD FACSAria III system acquisition rates

BD_FACSAria_III_brochure[1]

FSC-A

Differentiating H9(x 1,000)-226

SSEA-3 PE-ADifferentiating H9Differentiating H9

0102030405060708050100150Differentiating H901020304050607080Differentiating H9510105CountCount

SSEA-1 FITC-A4SSEA-3 PE-A10104P1P2CountP3010-42010102103104105SSEA-1 FITC-A

-2260102103104105SSEA-3 PE-A-10001010101033TRA-1-81 Alexa 647-A2100-226-420102-1000102103104105TRA-1-81 Alexa 647-A-100010101010TRA-1-81 Alexa 647-ADifferentiating 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

cells. This combination of markers has been widely used to characterize and isolate

differentiated and undifferentiated stem cells derived from hESCs and iPS cells.

14

BD_FACSAria_III_brochure[1]

BD_FACSAria_III_brochure[1]

Regional Offices /offices

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