BD FACSAria III Addendumto the BD FACSAria II User’s Guide · X-mount optical plate The fiber...

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bdbiosciences.com 648240 Rev. A 1/2010 For Research Use Only. Not for use in diagnostic or therapeutic procedures. Becton, Dickinson and Company BD Biosciences San Jose, CA 95131 Tel 877.232.8995 Fax 800.325.9637 [email protected] Asia Pacific Tel 65.6.861.0633 Fax 65.860.1593 Europe Tel 32.2.400.98.95 Fax 32.2.401.70.94 [email protected] Brazil Tel 55.11.5185.9995 Fax 55.11.5185.9895 [email protected] Japan Nippon Becton Dickinson Company, Ltd. Toll Free 0120.8555.90 Tel 81.24.593.5405 Fax 81.24.593.5761 Canada Toll Free 888.259.0187 Tel 905.288.6000 Fax 888.229.9918 [email protected] Mexico Toll Free 01.800.236.2543 Tel 52.55.5999 8296 Fax 52.55.5999.8288 [email protected] BD FACSAria III Addendum to the BD FACSAria II User’s Guide Including the 561-nm and 445-nm Laser Options

Transcript of BD FACSAria III Addendumto the BD FACSAria II User’s Guide · X-mount optical plate The fiber...

Page 1: BD FACSAria III Addendumto the BD FACSAria II User’s Guide · X-mount optical plate The fiber optic heads for the new lasers and all existing lasers (except the 375 nm) are mounted

bdbiosciences.com648240 Rev. A1/2010

For Research Use Only. Not for use in diagnostic or therapeutic procedures.

Becton, Dickinson and CompanyBD BiosciencesSan Jose, CA 95131Tel 877.232.8995Fax [email protected]

Asia PacificTel 65.6.861.0633Fax 65.860.1593

EuropeTel 32.2.400.98.95Fax [email protected]

BrazilTel 55.11.5185.9995Fax [email protected]

JapanNippon Becton Dickinson Company, Ltd.Toll Free 0120.8555.90Tel 81.24.593.5405Fax 81.24.593.5761

CanadaToll Free 888.259.0187Tel 905.288.6000Fax [email protected]

MexicoToll Free 01.800.236.2543Tel 52.55.5999 8296Fax [email protected]

BD FACSAria III Addendumto the BD FACSAria II

User’s GuideIncluding the 561-nm and 445-nm

Laser Options

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Copyrights and trademarks

© 2010, Becton, Dickinson and Company. All rights reserved. No part of this publication may be reproduced, transmitted, transcribed, stored in retrieval systems, or translated into any language or computer language, in any form or by any means: electronic, mechanical, magnetic, optical, chemical, manual, or otherwise, without prior written permission from BD Biosciences.

The information in this guide is subject to change without notice. BD Biosciences reserves the right to change its products and services at any time to incorporate the latest technological developments. Although this guide has been prepared with every precaution to ensure accuracy, BD Biosciences assumes no liability for any errors or omissions, nor for any damages resulting from the application or use of this information. BD Biosciences welcomes customer input on corrections and suggestions for improvement.

Cy™ is a trademark of Amersham Biosciences Corp. Cy™ dyes are subject to proprietary rights of Amersham Biosciences Corp and Carnegie Mellon University and are made and sold under license from Amersham Biosciences Corp only for research and in vitro diagnostic use. Any other use requires a commercial sublicense from Amersham Biosciences Corp, 800 Centennial Avenue, Piscataway, NJ 08855-1327, USA.

Alexa Fluor® and Texas Red® are registered trademarks and Pacific Blue™ is a trademark of Molecular Probes, Inc.

Living Colors® is a registered trademark of Clontech.

BD, BD Logo and all other trademarks are property of Becton, Dickinson and Company. © 2010 BD

FCC information

WARNING: Changes or modifications to this unit not expressly approved by the party responsible for compliance could void the user’s authority to operate the equipment.

NOTICE: This equipment has been tested and found to comply with the limits for a Class A digital device, pursuant to Part 15 of the FCC Rules. These limits are designed to provide reasonable protection against harmful interference when the equipment is operated in a commercial environment. This equipment generates, uses, and can radiate radio frequency energy and, if not installed and used in accordance with the instruction manual, may cause harmful interference to radio communications. Operation of this equipment in a residential area is likely to cause harmful interference in which case the user will be required to correct the interference at his or her own expense. Shielded cables must be used with this unit to ensure compliance with the Class A FCC limits. This Class A digital apparatus meets all requirements of the Canadian Interference-Causing Equipment Regulations. Cet appareil numérique de la classe A respecte toutes les exigences du Réglement sur le matériel brouilleur du Canada.

History

Revision Date Change made

648240 Rev. A 1/2010 New document

Page 3: BD FACSAria III Addendumto the BD FACSAria II User’s Guide · X-mount optical plate The fiber optic heads for the new lasers and all existing lasers (except the 375 nm) are mounted

Contents

About this addendum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

BD FACSAria III flow cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

561-nm and 445-nm laser options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Laser power switches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Laser and optics configurations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Detector arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Additional system configurations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Special considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Accessing the additional octagon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Setting up configurations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Typical workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Optics part numbers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

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About this addendum

About this topic This topic describes the purpose of this document and the intended audience.

Purpose The addendum serves multiple purposes.

This addendum covers the new features for the BD FACSAria™ III cell sorter. This addendum is intended to be used in conjunction with the BD FACSAria II User’s Guide.

This addendum is also for BD FACSAria and BD FACSAria II customers who have upgraded their systems with the 561-nm or 445-nm laser options. An upgraded BD FACSAria II becomes a BD FACSAria IIIu.

Features described The BD FACSAria III cell sorter includes the following features, even if the system does not receive a 561-nm or 445-nm laser option.

The X-mount optical plate

An optical alignment that includes a fourth beam spot for future upgrading to additional lasers

A BD FACSAria III flow cell

An octagon detector array for the 405-nm and 375-nm lasers

Safety The content of the BD FACSAria II Safety and Limitations Manual applies to the BD FACSAria III cell sorter. Make sure that everyone working with the system is familiar with the safety details in the Safety and Limitations Manual that is shipped with each system.

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BD FACSAria III Addendum to the BD FACSAria II User’s Guide2

BD FACSAria III flow cell

About this topic The topic describes the BD FACSAria III flow cell and the integrated nozzles that are used with it.

The BD FACSAria III system includes a new flow cell that has been optimized for four laser beam spots. This flow cell is also included with the 375-nm laser option for side population studies.

Integrated nozzles for the flow cell

The BD FACSAria III flow cell requires integrated nozzles that have been optimized for it. The system is shipped with these nozzles. These nozzles have also been optimized for BD FACSAria II systems equipped with a 375-nm laser. The following table lists the part numbers for the nozzles, in case you need to order replacement nozzles.

The nozzles can be identified by the MS marking on the bottom side of the handle. See the following figure.

The 130-micron and closed-loop nozzles are the same as those used in the BD FACSAria II system. See the user’s guide for those part numbers.

Nozzle size Part number

70 micron 647339

85 micron 647340

100 micron 647341

MS indicates integrated nozzle for the BD FACSAria III flow cell

Bottom view of nozzle

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561-nm and 445-nm laser options

About this topic This topic describes the laser options and the X-mount optical plate.

Either one or both of these lasers can be added at the factory as an option on a new instrument, or they can be installed by a BD field service engineer into an existing system at your facility.

561-nm laser option The 561-nm laser can be installed on a system to enable improvements in multicolor experiments and to expand the range of applications for these platforms. Specifically, this option increases PE sensitivity and minimizes or eliminates FITC spectral overlap into the PE channel, giving the instrument the best chance at resolving a dim staining PE population. In addition, this laser improves resolution for mCherry fluorescent proteins.

445-nm laser option The 445-nm laser can be installed on a system to enable enhanced sensitivity when working with Cyan fluorescent proteins. This laser shares the detector collection optics with the 488-nm laser. If both lasers are installed on a system, you can use either the 445-nm laser or the 488-nm laser, but you cannot use both at the same time. The laser power switches are designed to select one laser or the other.

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BD FACSAria III Addendum to the BD FACSAria II User’s Guide4

X-mount optical plate The fiber optic heads for the new lasers and all existing lasers (except the 375 nm) are mounted on an X-mount optical plate. The following figure shows the location for each fiber optic head.

Fourth beam spot A fourth spatially-separated beam spot is added with the 561-nm laser option. This additional beam spot enables the simultaneous use of four lasers.

Changes to laser delays If you are an existing BD FACSAria or BD FACSAria II customer, you will notice a difference in default laser delays with your 561-nm or 445-nm laser upgrade compared to your current system. This is due to a change in the laser order and the addition of a fourth laser beam spot.

The laser delays are set automatically when you use BD™ Cytometer Setup and Tracking (CS&T). If you need to set laser delays manually, see the procedure in your user’s guide.

445 nm

405 nm

488 nm

633 nm

561 nm

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Laser power switches

About this topic This topic describes the rotary laser power switches that are installed on the system for the laser options.

Power switch for 6-laser system

This figure shows the rotary switches in a 6-laser system. The switch labels and the functionality of the switches can vary depending on how many lasers are installed.

Using power switches You must turn on the lasers you need for an experiment by rotating the switches to the desired settings. Allow 30 minutes for the lasers to warm up.

MAIN POWER

488 ON

OFF

445 ON

405 ON

OFF

375 ON

OFF

633 ON

561 ON

561+633 ON

3-position switch

4-position switch

3-position switch

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BD FACSAria III Addendum to the BD FACSAria II User’s Guide6

Laser and optics configurations

About this topic This topic provides information about the laser configurations and the mirrors, filters, and fluorochromes required for each laser, depending on the application you are performing.

System configurations BD FACSAria III systems can be configured with up to six lasers, with four lasers active at one time. The following table contains typical configuration information for each laser. See Additional system configurations (page 13) for configurations with fewer than six lasers.

Laser and optics device PMTs LP mirrors BP filters Fluorochromes

Blue - 488 nm

Trigon

A 655 LP 695/40 675/20

PerCP-Cy™5.5PerCP

B 502 LP 530/30 FITC

C — 488/10 SSC

Purple - 445 nm

Trigon

A 470 LP 510/80 CFP, AmCyan fluorescent protein

B — 445/15 SSC

C - Optional

Yellow-green - 561 nm

Octagon

A 735 LP 780/60 PE-Cy™7

B 630 LP 670/14 710/50

PE-Cy™5PE-Cy5.5

C 600 LP 610/20 PE-Texas Red®, Living Colors® mCherry, PI

D — 582/15 PE

E, F - Optional

G, H - Not available

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Optics choices for 375-nm laser

The optics listed for the 375-nm laser in the previous table are specifically intended for side populations studies. For other experiments, you can use the optics from the 405-nm laser with the 375-nm laser.

Using a fluorochrome with multiple lasers

If you plan to use a fluorochrome with more than one laser, you must create a unique name for each instance. For example, in the previous table, PI is listed for the 561-nm laser on PMT C. Then it is listed as propidium iodide for the 375-nm laser on PMT B. You could also name them PI-561 and PI-375, or something similar, as long as the names are unique.

Red - 633 nm

Trigon

A 735 LP 780/60 APC-Cy7, APC-H7

B — 660/20 APC

C - Optional

Violet - 405 nm

Octagon

A 502 LP 510/50 BD Horizon™ V500, AmCyan

B — 450/40(480/20 if using 445 laser)

BD Horizon V450, Pacific Blue™, DAPI

C, D, E, F, G, H - Optional

Near UV - 375 nm

Octagon

A 600 LP 670 LP Hoechst Red, propidium iodide

B — 450/20 Hoechst Blue, DAPI

C, D, E, F, G, H - Optional

Laser and optics device PMTs LP mirrors BP filters Fluorochromes

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BD FACSAria III Addendum to the BD FACSAria II User’s Guide8

Detector arrays

About this topic This topic illustrates the layout of the detector arrays and the optical components in the standard octagons and trigons.

Detector array locations The following figure shows the locations of the detector arrays in a 6-laser system. The locations can differ depending on which lasers are installed in your system.

AC

B

AC

BA

C

E

G

B D

FH

AC

E

G

B D

FH

Front trigons and octagon

Back octagon

488/445-nm Trigon

633-nm Trigon

561-nm Octagon

405/375-nmOctagon

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561-nm laser detector array

The following drawing shows the mirrors and filters in a standard 561-nm laser detector array.

See Fifth PMT in the 561 octagon (page 15) for details on additional functionality when a fifth PMT is added to the 561-nm octagon.

AC

E

G

B D

FH

PE-Cy5

PE-Cy7

PE-TxRedmCherryPI

PE

735 LP600 LP

630 LP

780/60610/20

670/14 582/15

561-nm Octagon

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BD FACSAria III Addendum to the BD FACSAria II User’s Guide10

405-nm and 375-nm laser detector array

The following figure shows the mirrors and filters in a standard 405-nm laser detector array.

You can upgrade with additional PMTs for this array to add more capabilities to the system.

If the system has the optional 375-nm laser, then that laser shares the octagon with the 405-nm laser. Only one of the lasers can be active at a time.

V450 DAPI

V500 AmCyan

AC

EGB D

FH502 LP

510/50

450/40

405/375-nm Octagon

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488-nm and 445-nm laser detector array

The following drawing shows the mirrors and filters in the standard 488-nm and 445-nm laser detector arrays.

These two lasers share the same trigon. You must switch optics depending on which laser you are using. Only one of the lasers can be active at a time. The SSC changes from PMT C in the 448-nm trigon to PMT B in the 445-nm trigon.

See Optics change when using 445-nm laser (page 16) for details on switching optics in the 405/375-nm octagon when the 445-nm laser is being used.

PerCP-Cy5.5SSC

CFP

SSCFITC

AC

B

AC

B

655 LP470 LP

502 LP

695/40

530/30

510/80

445/15

488/10

488-nmTrigon

445-nm Trigon

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BD FACSAria III Addendum to the BD FACSAria II User’s Guide12

633-nm laser detector array

The following figure shows the mirrors and filters in a standard 633-nm laser detector array.

See Third PMT in the 633 trigon (page 16) for information on adding a third PMT to this trigon.

APC-Cy7APC-H7

APC

AC

B735 LP

780/60

660/20

633-nm Trigon

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Additional system configurations

About this topic This topic describes the configurations for systems with two, three, four, or five lasers installed.

System configurations with octagon locations

The following tables list the configurations that are available in addition to the 6-laser version. See the table on page 6 for the photomultiplier tube (PMT), optics, and fluorochrome assignments.

5-laser systems

4-laser systems

Lasers (nm) Front octagon Back octagon

488, 561, 633, 405, 375 405/375 561

488, 445, 561, 633, 405 405 561

488, 445, 561, 633, 375 375 561

488, 445, 633, 405, 375 488/445 405/375

Lasers (nm) Front octagon Back octagon

488, 561, 633, 405 405 561

488, 561, 633, 375 375 561

488, 445, 561, 633 561 None

488, 445, 633, 405 488/445 405

488, 445, 633, 375 488/445 375

488, 633, 405, 375 488 405/375

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BD FACSAria III Addendum to the BD FACSAria II User’s Guide14

3-laser systems

2-laser systems

Lasers (nm) Front octagon Back octagon

488, 561, 633 561 None

488, 445, 633 488/445 None

488, 633, 405 488 405

488, 633, 375 488 375

Lasers (nm) Front octagon Back octagon

488, 633 488 None

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Special considerations

About this topic This topic describes some optional configurations and special cases that should be considered, depending on your system.

Fifth PMT in the 561 octagon

A system with a 561-nm laser and an optional fifth PMT includes a 685 LP mirror that can be used with the supplied 710/50 BP filter to run PE-Cy5.5. The optics in the PMTs need to be shifted to use this setup. The following drawing shows the optics configuration for this case.

AC

E

G

B D

FH

PE-Cy5.5PE-Alexa Fluor® 700

PE-Cy5

PE-Cy7PE

PE-TxRedmCherryPI

735 LP630 LP

685 LP 600 LP

780/60670/14

710/50 610/20

582/1

5

561-nm Octagonwith 5th PMT

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BD FACSAria III Addendum to the BD FACSAria II User’s Guide16

Third PMT in the 633 trigon

If you upgrade with an additional PMT in the 633 trigon, the upgrade kit comes with an Alexa Fluor® 700 filter set, which contains 690 LP and 730/45 BP filters. This enables the simultaneous use of APC, Alexa Fluor® 700, and APC-H7 (Cy7).

Optics change when using 445-nm laser

In a system with a 445-nm laser turned on, the BP filter for the 405-nm laser must be changed to a 480/20 (from the standard 450/40). This is because the 405 detector picks up scattered light from the 445-nm laser and causes CS&T to produce incorrect results. This filter change applies to the 375-nm laser when using the same optics as the 405-nm laser.

Optional PMT upgrades You can order PMT upgrades for certain detector arrays to enable additional color capabilities for your system. For octagons located in the back, the maximum number of PMTs is six. Contact your BD Biosciences representative for additional information.

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17

Accessing the additional octagon

About this topic This topic describes how to gain access to the back octagon that is installed behind the main detector door. Access is required when you change the optics in the back octagon.

Procedure To access the back octagon:

1. Open the optics access door on the front of the instrument by pushing in the button so it pops out and then turning it to open the door.

2. Open the detector door.

– BD FACSAria II and III systems: Use a hex wrench to open the closure mechanism.

– BD FACSAria systems: Slide the latch mechanism to open the door.

o

Caution: Laser! Do not open the main laser cover. Doing so could expose you to laser radiation if the lasers are turned on.

Main laser cover. Do not open.

Closure mechanism for detector door

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BD FACSAria III Addendum to the BD FACSAria II User’s Guide18

3. Swing open the detector door. The new back octagon is installed behind the main detector door.

Back 561-nm octagon installed behind main detector door

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Setting up configurations

About this topic This topic describes the base configuration for the cytometer. It also describes how to create new custom configurations and delete invalid configurations.

Base configuration Your system has a base configuration that includes the number of lasers and the channel mapping of the PMTs.

Creating custom configurations

You must create custom configurations in CS&T for each cytometer setup you use for experiments. See the user’s guide for further information.

Deleting configurations If your system has been upgraded to include new lasers or PMTs, a new base configuration has been created. Previously existing cytometer configurations and application settings are no longer valid. A red exclamation mark indicates an invalid configuration. A blue circle indicates a valid configuration.

Invalid

Valid

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BD FACSAria III Addendum to the BD FACSAria II User’s Guide20

To delete existing invalid configurations:

1. Left-click to select the configuration, then right-click the configuration and select Delete.

The following dialog appears.

2. Select one of the following options:

– Delete Configuration Only. Deletes the configuration, but leaves the catalog, CS&T reports, Levey-Jennings data, and application settings.

– Delete Configuration and Setup Results. Deletes the configuration and all associated CS&T data. The CS&T reports and FCS files remain in the default directory, which is:

C:\ BDCytometerSetupAndTracking

If you have changed the default directory, then the files will be in the user-selected directory.

3. Click OK.

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21

Typical workflow

About this topic This topic describes the typical workflow for a system with new lasers installed.

Workflow Operation of the system with new lasers is essentially the same as with a standard system, with the addition of new capabilities offered by the new laser wavelengths. See the following table for an outline of a typical workflow.

Step Action

1 Turn on the main power and the specific lasers for the experiment you need to run. Allow the lasers to warm up for 30 minutes.

2 Start BD FACSDiva™ software and perform fluidics startup and set up the stream.

3 Ensure that the appropriate cytometer configuration is set for your application.

4 Run a performance check using CS&T beads, if needed.

5 Proceed with running experiments and sorting as described in the user’s guide.

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BD FACSAria III Addendum to the BD FACSAria II User’s Guide22

Optics part numbers

About this topic This topic lists the part numbers for the optics used on the system.

Part numbers for LP mirrors

The following table lists the part numbers for the longpass (LP) mirrors used in the system.

LP mirrors Part number LP mirrors Part number

470 LP 648920 670 LP 642328

502 LP 343797 685 LP 649617

600 LP 334925 690 LP 649615

630 LP 338810 735 LP 343787

655 LP 343789

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23

Part numbers for BP filters

The following table lists the part numbers for the bandpass (BP) filters used in the system.

BP filter Part number BP filter Part number

445/15 648921 610/20 334934

450/20 642329 660/20 343800

450/40 343801 670/14 334933

480/20 649139 675/20 343791

488/10 343799 695/40 343790

510/50 649421 710/50 649422

510/80 648922 730/45 649618

530/30 343798 780/60 343788

582/15 649046

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