BD FACSCanto II Reference Manual
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BD FACSCanto II Reference Manual

Flow cytometer
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For In Vitro Diagnostic Use
bdbiosciences.com
Part No. 640806 Rev. A
May 2006
BD Biosciences
Asia Pacific
2350 Qume Drive
Tel (65) 6-861-0633
San Jose, CA 95131-1807
Fax (65) 6-860-1590
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Tel (877) 232-8995
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BD FACSCanto II
Flow Cytometer
Reference Manual
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  • Page 1 BD FACSCanto II Flow Cytometer Reference Manual For In Vitro Diagnostic Use bdbiosciences.com Part No. 640806 Rev. A May 2006 BD Biosciences Asia Pacific Brazil Canada 2350 Qume Drive Tel (65) 6-861-0633 Tel (55) 11-5185-9995 Tel (888) 259-0187 San Jose, CA 95131-1807...
  • Page 2 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.
  • Page 3 BD Biosciences, it is the buyer/user’s responsibility to install and maintain up-to-date virus protection software. BD Biosciences does not make any warranty with respect to the workstation remaining virus free after installation. BD Biosciences is not liable for any claims related to or resulting from buyer/user's failure to install and maintain virus protection.

  • Page 5: Table Of Contents

    Computer Workstation ........BD FACS Loader (Optional) ....... . .
  • Page 6 Replacing the Fluidics Cart Fuses ......BD FACSCanto II Flow Cytometer Reference Manual...
  • Page 7 Chapter 5: Troubleshooting Instrument Troubleshooting ........Fluidics Cart Troubleshooting .
  • Page 8 Capacity ..........BD FACS Loader Specifications ........

  • Page 9: About This Manual

    BD Biosciences recommends that first-time users of this instrument take advantage of operator training offered with the sale of every new instrument. The BD FACSCanto II Flow Cytometer Reference Manual assumes you have a working knowledge of basic Microsoft® Windows® operation. If you are not familiar with the Windows operating system, refer to the documentation provided with your computer.

  • Page 10: Conventions

    For example, “select File > Print” means select Print from the File menu. Ctrl-X Press the indicated keys simultaneously For example, Ctrl-P means hold down the Control key while pressing the letter p. BD FACSCanto II Flow Cytometer Reference Manual...

  • Page 11: Technical Assistance

    • error messages • details of system performance For instrument support within the US, call (877) 232-8995. For support within Canada, call (888) 259-0187. Customers outside the US and Canada, contact your local BD representative or distributor. About This Manual...
  • Page 12 BD FACSCanto II Flow Cytometer Reference Manual...

  • Page 13: Chapter 1: Introduction

    The instrument does not require special facilities—it plugs into a standard wall outlet, uses air-cooled lasers, and provides its own air pressure and vacuum for the fluidics and waste. You can prepare samples on the BD FACS™ Sample Prep Assistant II and import the sample prep worklist.

  • Page 14: Intended Use

    Intended Use The BD FACSCanto II system is intended for use as an In Vitro Diagnostic device for identification and enumeration of lymphocyte subsets in human cells in suspension for flow cytometry. BD FACSCanto II Flow Cytometer Reference Manual...

  • Page 15: System Components

    System Components The BD FACSCanto II system consists of a benchtop flow cytometer, a self- contained fluidics cart, and the BD FACSCanto II workstation. System options include an automated sample loader and a barcode reader. For a description of system components, see: •...

  • Page 16: Flow Cytometer

    The BD FACSCanto II flow cytometer consists of a fluidics subsystem, an optics subsystem, and an electronics subsystem. For a more in-depth discussion of fluidics, optics, electronics, and flow cytometry, see the Technical Overview on page 105.
  • Page 17 flow cell tubing tube sensor Bal seal access tube eject cylinder (used with Loader option only) tube guide sample injection tube (SIT) (Loader option only) aspirator arm aspirator arm bar The following table briefly describes these components. flow cell where laser beam intercepts particles tubing brings sheath and cleaning fluids to and aspirates waste from flow cell hollow metal tube that brings sample to flow cell...
  • Page 18 The flushed sheath solution is aspirated by the aspirator arm. SIT cleaning between tubes is automatic when you use BD FACSCanto clinical software. In BD FACSDiva software, SIT cleaning between tubes is automatic unless you disable it by deselecting the SIT Flush checkbox on the Acquisition Dashboard.
  • Page 19 Optics The optics system in the BD FACSCanto II cytometer is composed of both excitation optics and collection optics. Excitation optics bring light to the flow cell. Collection optics gather the light signals emitted or scattered by the particles.
  • Page 20 The excitation optics consist of lasers, fiber optic cables, beam-shaping prisms, and an achromatic focusing lens, as shown in Figure 1-3. The BD FACSCanto II instrument uses low-powered air-cooled and solid state lasers that do not have special power and cooling requirements.
  • Page 21 Collection Optics From the flow cell, laser light is routed to the collection optics, which efficiently gather the signals emitted and scattered from each particle. The 4-2 configuration collection optics include two detector arrays, which consist of photomultiplier tubes (PMTs) arranged in one octagon and one trigon as shown in Figure 1-4. The octagon contains five PMTs and detects light from the 488-nm (blue) laser.
  • Page 22 The obscuration bar prevents excess laser light from entering this diode (see Figure 1-2 on page 19). For additional descriptions of detector arrays, mirrors, and filters, see Optics System on page 109. BD FACSCanto II Flow Cytometer Reference Manual...
  • Page 23 Some of the collection optics can be viewed by opening the optics access door. The 4-2 configuration octagon and trigon arrays have the filter and mirror combinations shown in Table 1-2. Table 1-2 Octagon and trigon optical filters (4-2 configuration) Detector Array BP Filter or LP Mirror...
  • Page 24 BD FACSCanto II electronic system components consist of power controls, connectors, the electronics processing board, the fluidics controllers, and the acquisition indicator lights. This section describes only the user-accessible power panel and the acquisition indicator lights.
  • Page 25 Acquisition Indicator Lights The acquisition indicator lights are located on the flow cell access cover on the front of the cytometer (see Figure 1-1 on page 16). Each light corresponds to a detector in the collection optics subsystem, and blinks when the signal at that detector reaches a preset level (acquisition threshold levels override the presets).

  • Page 26: Fluidics Cart

    (although the cart can be hooked up to an in-house air source). Figure 1-7 Fluidics cart cubitainer 10-L waste container door access knob condensation trap BD FACSCanto II Flow Cytometer Reference Manual...
  • Page 27 Containers and Ports The fluidics cart holds a 10-L waste container, a 20-L BD FACSFlow™ cubitainer, a 5-L BD FACS shutdown solution cubitainer, and a 5-L BD FACSClean solution cubitainer. Use the waste container provided with the system. Do not substitute other containers.
  • Page 28 Leave the auxiliary air supply switch off unless the cart has been attached to an in-house air supply by BD Biosciences service personnel (see Table 1-3 for details). Leave the cart circuit breaker on at all times.

  • Page 29: Computer Workstation

    BD FACS Loader (Optional) The BD FACS Loader automatically introduces prepared samples to the cytometer. The Loader is controlled by BD FACSCanto clinical software or BD FACSDiva software. It can be added to your system at any time. The Loader consists of: •...

  • Page 30: Barcode Reader (Optional)

    The Opticon LG2 Imager is a hand-held barcode reader that plugs into the USB port on the BD FACSCanto II computer workstation. A green light on the top of the barcode reader indicates that it is connected to the USB port and ready to use.

  • Page 31: System Requirements

    Both included software packages must be installed: • BD FACSCanto II clinical software v2.1 Do not read FCS files created with v2.0 or v2.1 into previous versions of BD FACSCanto II clinical software. Previous versions will show incorrect results. • BD FACSDiva software v5.0...
  • Page 32 Fluids Required for External Cleaning • BD FACSClean solution • Deionized (DI) water Setup Beads BD FACS 7-color setup beads for use with BD FACSCanto clinical software BD FACSCanto II Flow Cytometer Reference Manual...

  • Page 33: Chapter 2: Bd Facs Loader Option

    BD FACS Loader Option This chapter contains the following information: • Loader Components on page 34 • Using the Loader on page 36 • Maintaining the Loader on page 42...

  • Page 34: Loader Components

    (SIT) BD FACSCanto II Flow Cytometer Reference Manual...
  • Page 35 Not all manufactured 12 x 75-mm tubes have been checked for proper functionality on the Loader. BD Biosciences has validated only disposable, 12 x 75-mm BD Falcon polystyrene test tubes, BD Trucount tubes, and BD FACS 7-color setup bead tubes.

  • Page 36: Using The Loader

    The Loader operates by software commands only. Commands are issued with either BD FACSCanto clinical software or BD FACSDiva software. For instructions on running samples with the Loader, refer to the BD FACSCanto II Instructions for Use. Opening and Closing the Loader Doors Sliding doors are located in front of and to the right of the Loader drawer.

  • Page 37: Acquiring With The Loader

    The tube guide should be forward, and the aspirator arm bar should be horizontal and aimed to the front (toward the carousel). Figure 2-3 Automatic loading setup tube guide aspirator arm bar For detailed setup instructions, see Changing to Automatic Loading on page 41. Chapter 2: BD FACS Loader Option...
  • Page 38 For accurate results, match the tubes to those listed on the printed worklist (BD FACSCanto clinical software) or the Carousel Assignment tab (BD FACSDiva software). Print a copy of tube assignments and use the printout as a guide when filling each carousel.
  • Page 39 To run the Loader, the cover doors must be closed. Tubes will not be loaded if the cover doors are open. The currently running tube will be unloaded if the cover doors are opened during a run. Loader cover doors (closed) Chapter 2: BD FACS Loader Option...

  • Page 40: Changing Operational Modes

    Pull out the drawer. Remove any carousel in the Loader. In manual mode, you do not need the tube guide arm. Move it all the way back and away from you. automatic mode manual mode BD FACSCanto II Flow Cytometer Reference Manual...
  • Page 41 Wear suitable protective clothing, eyewear, and gloves. If the aspirator arm bar is not already in the forward position, rotate it to a horizontal position and aimed to the front (toward the carousel). manual mode automatic mode Chapter 2: BD FACS Loader Option...

  • Page 42: Maintaining The Loader

    Spill Containment Area The top of the Loader has a depressed area intended to contain spills that may occur during operation. Check this area for fluids and clean as described in the following section. BD FACSCanto II Flow Cytometer Reference Manual...

  • Page 43: Cleaning External Surfaces

    Switch off the cytometer power and unplug the AC power cord from the wall socket. Wipe all accessible surfaces with BD FACSClean solution. Ensure that the optical sensors are cleaned. Saline deposits can develop on the sensors, causing tubes in the carousel to be missed.
  • Page 44 BD FACSCanto II Flow Cytometer Reference Manual...

  • Page 45: Chapter 3: Barcode Reader Option

    Barcode Reader Option This chapter contains the following information: • Installing and Using the Barcode Reader on page 46 • Cleaning the Barcode Reader on page 48 • Barcode Symbologies on page 49...

  • Page 46: Installing And Using The Barcode Reader

    BD FACSCanto II is capable of reading both 1D and 2D barcode symbologies. See Barcode Symbologies on page 49. The BD FACSCanto II system supports only two uses of the 2D barcode reader: • reading information from the BD FACS 7-color setup beads label into BD FACSCanto clinical software (2D barcode symbology) •...
  • Page 47 Patient information is scanned from sample labels into the worklist ID field. Locate the barcode on the setup values label of BD FACS 7-color setup beads kit, or on the patient sample. For accurate results, do not photocopy or enlarge the barcodes that are included with the reagent.

  • Page 48: Cleaning The Barcode Reader

    • isopropyl alcohol • ethyl alcohol (denatured grade) Do not use BD FACSClean solution (or bleach) to clean or disinfect the barcode reader. BD FACSCanto II Flow Cytometer Reference Manual...

  • Page 49: 1D Barcode Symbologies

    By default, the barcode reader has checksums enabled. We recommend you do not disable checksums, or use barcode symbologies without checksums. 1D Barcode Symbologies BD Biosciences has evaluated the following 1D barcode symbologies for use with the BD FACSCanto II flow cytometer, and has these recommendations: Barcode Symbology...

  • Page 50: 2D Barcode Symbologies

    2D Barcode Symbologies BD Biosciences has evaluated 2D barcode symbology to read the target values of BD FACS 7-color setup beads when using BD FACSCanto clinical software. 2D barcode symbology is required to read all target values with one scan.

  • Page 51: Chapter 4: Maintenance

    Maintenance The BD FACSCanto II system requires basic preventive maintenance to preserve instrument reliability. This section explains the procedures you should follow to keep your instrument in good condition. • Scheduled Maintenance on page 52 • Unscheduled Maintenance on page 66...

  • Page 52: Scheduled Maintenance

    Purge fluid filters Removes air from fluid filters, Weekly ensuring they will not dry out See Purging the Fluidics Filters on page 57. Change the waste tank See step 6 on page 56. Monthly BD FACSCanto II Flow Cytometer Reference Manual...
  • Page 53 Decontaminates the internal Monthly and before (Long Clean) sheath path with service calls BD FACSClean solution, then rinses with BD FACS shutdown solution See Decontaminating the Fluidics System (Long Clean) on page 59. Replace the air filter Ensures proper instrument...

  • Page 54: Emptying The Waste Container

    (10% of total volume) before disposal. Dispose of waste in accordance with local regulations. Use proper precaution and wear suitable protective clothing, eyewear, and gloves. waste port disposable waste cap trap waste tank BD FACSCanto II Flow Cytometer Reference Manual...
  • Page 55 Ensure the cytometer is not acquiring events. Detach the waste container’s sensor and fluid line from the fluidics cart waste port. • Pull the sensor straight out. • Press the metal clip on the fluid line. waste fluid line with metal clip sensor The waste tank can become pressurized when the cytometer is running.
  • Page 56 Tighten each component only until it is hand-tight. Do not use sealants such as Teflon® tape or other adhesives. Screw the cap assembly onto the tank and hand-tighten until it is fully closed. Re-attach the sensor and fluid lines. BD FACSCanto II Flow Cytometer Reference Manual...

  • Page 57: Purging The Fluidics Filters

    Purging the Fluidics Filters Once a week, purge air from all fluid filters by opening the bleeder valve on the top of each filter, one at a time. This ensures that the filters will not dry out. Prime fluid lines before purging the filters, as noted in the procedure. The cytometer must be on (system pressurized) during this procedure.
  • Page 58 If the cubitainer contains fluid and the fluid lines are attached and primed, the filter might be airlocked. See Removing an Air Lock on page 72. Close the valve by turning it clockwise. Repeat steps 2 through 5 with the remaining filters. BD FACSCanto II Flow Cytometer Reference Manual...

  • Page 59: Decontaminating The Fluidics System (Long Clean)

    The Cytometer menu in BD FACSCanto clinical software differs slightly from the BD FACSDiva Instrument menu. Instructions in this and following sections list the BD FACSCanto II menu first, followed by the BD FACSDiva menu, in parentheses. Check all fluid levels. Empty the waste if needed.

  • Page 60: Replacing The Air Filter

    If setup fails after a long clean, repeat Fluidics Startup until setup passes. Replacing the Air Filter The BD FACSCanto II instrument has an air filter located in the side door. Replace the filter every 6 months to ensure proper instrument performance. BD FACSCanto II Flow Cytometer Reference Manual...
  • Page 61 Materials replacement filter (see Other Replacement Parts on page 141 for ordering) Procedure Turn off the cytometer power. Open the side door: • Press the black button—a handle will pop out. • Twist the handle and pull. Chapter 4: Maintenance...
  • Page 62 Install a new filter. Ensure that the arrows etched on edges of the new filter point in toward the instrument (arrows indicate direction of air flow). Turn the tabs to hold the filter in place. BD FACSCanto II Flow Cytometer Reference Manual...

  • Page 63: Replacing Fluidics Filters

    Close the side door: • Twist the handle while closing door. • Push door closed until the door latches. • Push the handle in to latch the handle. To avoid instrument damage, do not close the door on any tubing or wires. Replacing Fluidics Filters Change the fluid filters when you see increased debris in FSC vs SSC plots, or every 6 months.
  • Page 64 Place a few paper towels beneath the filter to collect drips. Remove the filter by pressing the tabs on each quick-disconnect coupling. Figure 4-2 Removing the filter metal tab loosen this bleeder valve FLOW quick-disconnect coupling metal tab BD FACSCanto II Flow Cytometer Reference Manual...
  • Page 65 Position the new filter and connect the couplings. Write today’s date on the filter so you will know when to replace it. Loosen the top bleeder valve by turning it counterclockwise. NOTICE Do not loosen the bottom bleeder valve. Ensure it is tightened. loosen this bleeder valve DO NOT loosen this...

  • Page 66: Unscheduled Maintenance

    See Cleaning the Flow Cell on needed page 76. Bubble Filter Purge Removes air from the bubble If fluidics run dry, or filter when CVs are poor See Purging the Bubble Filter on page 78. BD FACSCanto II Flow Cytometer Reference Manual...
  • Page 67 Description and Location When to Perform Decontaminate the Cleans out the fluidics lines with Before long-term storage fluidics system for BD FACSClean solution, then storage fills them with BD FACS shutdown solution See Decontaminating the Fluidics System for Storage on page 79.

  • Page 68: Changing A Cubitainer

    Container Port Label Port Color waste Waste (A) orange BD FACSFlow solution BD FACSFlow blue BD FACS shutdown solution BD FACS Shutdown Solution yellow BD FACSClean solution BD FACSClean Solution yellow BD FACSCanto II Flow Cytometer Reference Manual...
  • Page 69 Procedure Ensure the cytometer is not acquiring events. Detach the sensor and fluid line from the cart. • Pull the sensor straight out. sensor • Press the metal clip on the fluid line. metal clip You could damage the sensor line if you leave it connected while changing a cubitainer.
  • Page 70 To ensure that the appropriate solutions are dispensed, match the label on the container to the port on the fluidics cart. Prime the fluidics. Important: Continue with the procedure in Priming Fluidics Lines on page 71. BD FACSCanto II Flow Cytometer Reference Manual...
  • Page 71 Choose Cytometer (Instrument) > Cleaning Modes > Prime After Tank Refill. Select the checkboxes for the cubitainers you changed; click OK. BD FACSCanto clinical software BD FACSDiva software Click OK when the completion message displays. BD FACSDiva software...

  • Page 72: Removing An Air Lock

    • bypass tubing Procedure Place a few paper towels beneath the air-locked filter to collect drips. Remove the filter by pressing the metal tabs. Figure 4-3 Removing a filter metal tab metal tab BD FACSCanto II Flow Cytometer Reference Manual...
  • Page 73 Choose Cytometer (Instrument) > Cleaning Modes > Prime After Tank Refill. Select the checkbox that corresponds to the filter you have bypassed. Click OK. BD FACSDiva software BD FACSCanto clinical software When the prime finishes, remove the bypass tubing. Reattach the filter to the fluidics cart. Chapter 4: Maintenance...

  • Page 74: Cleaning External Surfaces

    Figure 4-5 on page 80 and steps 3, 4, 9, and 10 of the Replacing the Bal Seal procedure that starts on page 79. NOTICE Do not use BD FACSClean solution or bleach to clean or disinfect the barcode reader. See Cleaning the Barcode Reader on page 48. BD FACSCanto II Flow Cytometer Reference Manual...
  • Page 75 To avoid potential shock, always switch off the power and unplug the AC power cord before you begin cleaning. Wipe all accessible surfaces with BD FACSClean solution. Wet a fresh cloth with DI water and wipe all bleach-exposed surfaces to prevent corrosion.

  • Page 76: Removing Bubbles From The Flow Cell

    If you still see bubbles, repeat the process. Cleaning the Flow Cell Use the Clean Flow Cell command to run a tube of BD FACSClean solution through the SIT and flow cell. After the procedure is complete, the cleaning fluid remains in the SIT and flow cell until you run fluidics startup or shutdown.
  • Page 77 A progress message is displayed during the cleaning. BD FACSDiva software BD FACSCanto clinical software After the completion message displays, wait 5 minutes. Waiting allows BD FACSClean solution to dissolve deposits in the flow cell cuvette. Click OK. Remove the tube from the SIT.

  • Page 78: Purging The Bubble Filter

    In BD FACSCanto clinical software, select Cytometer > Cleaning Modes > Bubble Filter Purge. • In BD FACSDiva software, select Instrument > Cleaning Modes > Bubble Filter Purge and Degas Flow Cell. Wait while the purge finishes. BD FACSDiva software BD FACSCanto clinical software Click OK when the completion message displays.

  • Page 79: Decontaminating The Fluidics System For Storage

    Decontaminating the Fluidics System for Storage If your cytometer is to be taken out of operation, perform the following procedure to clean out the fluidics lines and fill them with BD FACS shutdown solution. Perform steps 1 through 4 in Decontaminating the Fluidics System (Long Clean) on page 59.
  • Page 80 Figure 4-5 Bal seal and retainer Bal seal retainer Materials • proper protective equipment • Bal seal removal tool use this end to remove Bal seal • replacement Bal seal • DI water BD FACSCanto II Flow Cytometer Reference Manual...
  • Page 81 Procedure Turn off the cytometer. If you have a Loader installed: • Open the Loader cover doors. • Pull out the drawer. • If needed, remove the carousel. • Move the tube guide to manual position. See Changing to Manual Loading on page 40.
  • Page 82 Lower the retainer down the SIT and let it rest on the aspirator arm. Access the Bal seal at the notch on the right side of the SIT assembly. Unseat the Bal seal by using the Bal seal removal tool to gently pull it downward. BD FACSCanto II Flow Cytometer Reference Manual...
  • Page 83 Allow the Bal seal to slide down the SIT and rest on top of the retainer. Bal seal Center the Bal seal on the retainer. With one hand, support the retainer and Bal seal and with the other hand, move the aspirator arm to the left. Lower the retainer and Bal seal from the SIT.
  • Page 84 Use both hands to gently push it into its seated position. If the Bal seal is not quite in its seated position, replacing the retainer will seat it correctly. Bal seal, spring side up BD FACSCanto II Flow Cytometer Reference Manual...

  • Page 85: Resetting The Cytometer Circuit Breaker

    Rinse the retainer in DI water before replacing it on the SIT. Reinstall the retainer over the SIT and tighten in the direction shown. Test the installation by manually loading a tube onto the SIT and running fluid. If you are using a Loader, ensure that the system is ready for automatic loading.

  • Page 86: Reconnecting The Ethernet And Rs232 Cables

    Should any plugs, cords, or tubings become accidentally disconnected, use the following diagrams to reconnect the fluidics cart to the cytometer (Figure 4-6 on page 87). The ports and connectors are color coded. BD FACSCanto II Flow Cytometer Reference Manual...
  • Page 87 Figure 4-6 Cytometer and fluidics cart ports cytometer ports fluidics cart ports Cytometer ports and their corresponding fluidics cart ports are listed in Table 4-1. For example, ensure the tubing for f. Sheath (B) connects to the port labeled i. Fluid Out. See Table 4-2 on page 88 for port functions. Table 4-1 Correspondence of cytometer ports to fluidics cart ports Port or Button on Cytometer Port on Fluidics Cart...
  • Page 88 Auxiliary Air In There will be no tubing on this port unless connected to house air. m. Air Out Sends compressed air to cytometer n. Waste Waste in (non-aspirated) o. Waste Waste in (aspirated) BD FACSCanto II Flow Cytometer Reference Manual...

  • Page 89: Replacing The Fluidics Level Sensors

    Replacing the Fluidics Level Sensors Replace the fluidics level sensors when instructed to do so by a BD Biosciences service representative. Before you replace a sensor, try rinsing it with DI water. If you are changing the sensor on the waste tank, use proper precaution and wear suitable protective clothing, eyewear, and gloves.
  • Page 90 Ensure that the cytometer is not acquiring events. Detach the sensor and fluid line from the cart. Unscrew the cap on the cubitainer. Remove the level sensor assembly and discard into a suitable receptacle. BD FACSCanto II Flow Cytometer Reference Manual...
  • Page 91 Put a new level sensor assembly on the cubitainer or tank. Hand-tighten the cap until it is fully closed. Ensure use of the correct assembly (Figure 4-7). Figure 4-7 Level sensor assemblies waste level sensor BD FACSFlow level sensor BD FACSClean solution and (red connector) (blue connector) BD FACS shutdown solution level sensor (yellow connector) Reattach the sensor line and fluidics lines.

  • Page 92: Replacing The Fluidics Cart Fuses

    Removing the plug allows easier access to the fuse door. Insert a small screwdriver into the slot and gently pry the fuse door open. slot for screwdriver fuse door circuit breaker empty socket, plug removed BD FACSCanto II Flow Cytometer Reference Manual...
  • Page 93 Gently pry out the fuse drawer until you can grip it. fuse drawer fuse door Remove the fuse drawer, and remove and dispose of the old fuses. Note the positions of the old fuses before you remove them. Duplicate these positions with the new fuses. Chapter 4: Maintenance...
  • Page 94 Slide the drawer back into the instrument until it snaps into place. Close the fuse access door. Reconnect the power cord to the fluidics cart. Plug the cytometer power cord into the wall outlet and switch on the power. BD FACSCanto II Flow Cytometer Reference Manual...

  • Page 95: Chapter 5: Troubleshooting

    Clinical Software Reference Manual, the BD FACSDiva Software Reference Manual, or the BD FACSCanto II Instructions for Use. If you need additional assistance, contact BD Biosciences. Refer to our website, bdbiosciences.com, for up-to-date contact information. Troubleshooting suggestions can be found under the following topics: •...

  • Page 96: Instrument Troubleshooting

    Air in BD FACSFlow filter Purge air from the filter. See (fluidics cart) Purging the Fluidics Filters on page 57. Air lock in BD FACSFlow See Removing an Air Lock on filter (on fluidics cart) page 72. BD FACSCanto II Flow Cytometer Reference Manual...
  • Page 97 Bal seal improperly installed Reinstall or replace the Bal seal. or worn See Replacing the Bal Seal on page 79. Air lock in BD FACSFlow See Removing an Air Lock on filter page 72. Tube partially removed from Once you start to remove a tube...
  • Page 98 Instrument circuit breaker Switch on the cytometer circuit tripped breaker, followed by the instrument power (if off). Blown fuse in cytometer Turn off the instrument main controller power and call BD Biosciences. BD FACSCanto II Flow Cytometer Reference Manual...
  • Page 99 Use only uncapped 12 x 75-mm sample tube does not fit BD Falcon polystyrene test tubes, snugly on SIT BD Trucount tubes, and BD FACS 7-color setup beads tubes. See System Requirements on page 31. Defective or cracked tube • Transfer sample to new tube.
  • Page 100 Call BD Biosciences. Waste line to fluidics cart Turn off the cytometer power. disconnected Clean up the liquid, using proper precautions. Check that waste lines are securely plugged in. Turn on the cytometer power. BD FACSCanto II Flow Cytometer Reference Manual...

  • Page 101: Fluidics Cart Troubleshooting

    Clean up the liquid. Check and close all bleeder valves. See Purging the Fluidics Filters on page 57. Broken fluid line Contact BD Biosciences. Fluidics cart will not Cart circuit breaker off Switch on the cart circuit breaker. power on, cytometer on Auxiliary air supply on, cart Toggle the auxiliary air supply off.

  • Page 102: Loader Troubleshooting

    Bubble in flow cell diverts Degas flow cell. See Removing stream Bubbles from the Flow Cell on page 76. Communication error Restart the system. Perform fluidics startup. See BD FACSCanto II Instructions for Use. BD FACSCanto II Flow Cytometer Reference Manual...
  • Page 103 See step 4 on page 39. If the problem persists, contact BD Biosciences for assistance. Aspirator arm bar vertical Rotate the bar horizontally. See Changing to Automatic Loading on page 41.
  • Page 104 BD FACSCanto II Flow Cytometer Reference Manual...

  • Page 105: Appendix A: Technical Overview

    Appendix A Technical Overview This appendix provides more information about these topics. • Flow Cytometry on page 106 • Fluidics System on page 106 • Optics System on page 109 • Electronics System on page 119...

  • Page 106: Flow Cytometry

    Sheath Cubitainer to Flow Cell On the BD FACSCanto II flow cytometer, a separate fluidics cart houses the sheath cubitainer, cleaning fluid cubitainers, and the waste tank. Positive-pressure pumps in the cart send sheath fluid past a 0.22-µm filter to a pressurized interior reservoir within the cytometer, called the plenum.
  • Page 107 Figure A-1 Sheath and sample flow pathways for the BD FACSCanto II cytometer Sheath Flow Sample Flow sheath tank tube sheath filter interior reservoir (plenum) sample injection tube (SIT) bubble filter flow cell interrogation point waste tank You can view the current sheath fluid pressure in BD FACSCanto clinical software by displaying the Status window.
  • Page 108 (10 µL/min) (120 µL/min) sheath fluid sheath fluid sheath fluid sheath fluid sample sample When using BD FACSCanto clinical software, the cytometer automatically regulates the sample pressure according to the currently selected panel. BD FACSCanto II Flow Cytometer Reference Manual...

  • Page 109: Optics System

    This brief flash of light is collected, filtered, and then converted into an electrical signal by the detectors. Figure A-3 Light pathway through the BD FACSCanto II flow cytometer stained obscuration...

  • Page 110: Light Scatter

    FSC—indicates relative differences in the size of the cells or particles • SSC—indicates relative differences in the internal complexity or granularity of the cells or particles Figure A-4 Forward scatter (FSC) and side scatter (SSC) side scatter laser forward scatter BD FACSCanto II Flow Cytometer Reference Manual...

  • Page 111: Fluorescence

    Fluorescence When cells or particles stained with fluorochrome-conjugated antibodies or other dyes pass through a laser beam, the dyes can absorb photons (energy) and be promoted to an excited electronic state. In returning to their ground state, the dyes release energy, most of which is emitted as light. This light emission is known as fluorescence.

  • Page 112: Optical Filters

    Figure A-6 Effect of an optical filter on incident photons photons absorbed photons photons reflected transmitted Two kinds of filters are used on the BD FACSCanto II flow cytometer (default configuration): • longpass (LP) • bandpass (BP) Longpass Filter In general, LP filters (mirrors) pass wavelengths longer than the filter rating. For...
  • Page 113 Figure A-7 Longpass filter longpass wavelength (nm) Not all light with shorter wavelengths is absorbed or reflected. Some will still pass through. Bandpass Filter A BP filter transmits a relatively narrow range or band of light. Bandpass filters are typically designated by two numbers. The first number indicates the center wavelength and the second refers to the width of the band of light that is passed.

  • Page 114: Detectors

    Detectors convert light signals into electrical signals that can be processed by the electronics system and a computer and then displayed on a plot. There are two types of signal detectors in the BD FACSCanto II flow cytometer: the photodiode and the photomultiplier tube (PMT). The photodiode is used to detect the stronger FSC signal (generated by light from the blue laser).
  • Page 115 Figure A-9 Light pathway around an octagon (4-2 configuration) 670-735 nm 564–606 nm 515–545 nm 750–810 nm Behind the LP mirror, a 780/60 BP filter admits light from 750 to 810 nm and substantially blocks other wavelengths. The light that finally reaches PMT A will be from dyes such as PE-Cy7 that emit in this range.
  • Page 116 The beam continues around the array, with different light spectrum ranges collected at successive PMTs. Side scatter signals (blue laser light that was deflected by the internal irregularities of a particle) are collected at PMT F. BD FACSCanto II Flow Cytometer Reference Manual...

  • Page 117: Spillover

    Spillover The arrangement of filters and mirrors in the BD FACSCanto II enables each PMT to receive signals predominantly from a specific fluorochrome. However, fluorochromes emit light over a range of wavelengths. As a result, a signal from one fluorochrome can appear in a detector used for another fluorochrome.
  • Page 118 The software automatically determines these compensations during setup, which you can also manually adjust: • In BD FACSCanto clinical software, adjust spectral overlap on the Spectral Overlap tab. • In BD FACSDiva software, adjust spectral overlap in the Compensation tab in the Instrument frame.

  • Page 119: Electronics System

    Once compensation has been set for one sample, the spectral overlap setting (compensation value) remains valid whether a dim or bright sample is run. Figure A-14 illustrates this principle. Although the signals differ in intensity, the percentage of signal detected in the FITC and PE channels remains constant. Figure A-14 Two FITC signals of different intensity FITC different intensity FITC signals...

  • Page 120: Pulses

    The peak intensity, or height of the pulse, is measured at this point. As the particle leaves the beam, the pulse trails off. BD FACSCanto II Flow Cytometer Reference Manual...

  • Page 121: Pulse Measurements

    Pulse Measurements The pulse travels from the PMTs to the electronics board within the cytometer. Figure A-16 Electronics board FSC diode preamp preamp preamp preamp preamp preamp preamp preamp preamp preamp digital digital digital digital digital digital digital digital digital digital delay delay...
  • Page 122 • The sum of all signal intensity measurements that occur within a discrete time period becomes the pulse area. After height and area calculations occur, they are sent to the signal processors. BD FACSCanto II Flow Cytometer Reference Manual...
  • Page 123 The electronics board includes an embedded computer that sends pulse measurements and other data from the cytometer to your workstation. For more about digital theory, refer to Appendix B in the BD FACSDiva Software Reference Manual. For an in-depth discussion, visit our website at bdbiosciences.com/immunocytometry_systems/ and download the BD FACSDiva...
  • Page 124 BD FACSCanto II Flow Cytometer Reference Manual...

  • Page 125: Appendix B: Optics Configurations

    Appendix B Optics Configurations This appendix describes the components for each of the BD FACSCanto II optics configurations. • 4-2 Optics Configuration on page 126 • 4-2-2 Optics Configuration on page 130 • 5-3 Optics Configuration on page 134...

  • Page 126: 4-2 Optics Configuration

    Excitation Optics The BD FACSCanto II instrument excitation optics consist of lasers, fiber optic cables, beam-shaping prisms, and an achromatic focusing lens, as shown in Figure 1-3 on page 20. The 4-2 configuration uses low-powered air-cooled and solid state lasers that do not have special power and cooling requirements.
  • Page 127 Table B-1 4-2 configuration optical filters Detector Array BP Filter or LP Mirror Intended Dye (Laser) Position LP Mirror Octagon 780/60 PE-Cy7 (488-nm blue laser) PerCP-Cy5.5 or PerCP blank — optical holder 585/42 530/30 FITC blank 488/10 optical holder and pinhole blank blank —...
  • Page 128 Figure B-1 4-2 configuration octagon detector array 488 nm blue laser BD FACSCanto II Flow Cytometer Reference Manual...
  • Page 129 Figure B-2 4-2 configuration trigon detector array 633 nm red laser Appendix B: Optics Configurations...

  • Page 130: 4-2-2 Optics Configuration

    NOTICE The BD FACSCanto II system is cleared for use with six-color assays. The development of seven- and eight-color assays on this device will require validation prior to use as a diagnostic procedure.
  • Page 131 The 4-2 configuration octagon and trigon arrays have the filter and mirror combinations as shown in Table B-2, Figure B-3 on page 132, and Figure B-4 on page 133. Table B-2 4-2-2 configuration optical filters Detector Array BP Filter or LP Mirror Intended Dye (Laser)
  • Page 132 Figure B-3 4-2-2 configuration octagon detector array 488 nm blue laser BD FACSCanto II Flow Cytometer Reference Manual...
  • Page 133 Figure B-4 4-2-2 configuration trigon detector arrays 405 nm violet laser 633 nm red laser Appendix B: Optics Configurations...

  • Page 134: 5-3 Optics Configuration

    NOTICE The BD FACSCanto II system is cleared for use with six-color assays. The development of seven- and eight-color assays on this device will require validation prior to use as a diagnostic procedure.
  • Page 135 Table B-3 5-3 configuration optical filters Detector Array BP Filter or LP Mirror Intended Dye (Laser) Position LP Mirror Octagon 780/60 PE-Cy7 (488-nm blue laser) PerCP-Cy5.5 or PerCP ® 616/23 PE-Texas Red 585/42 530/30 FITC blank 488/10 optical holder and pinhole blank blank —...
  • Page 136 Figure B-5 5-3 configuration octagon detector array 488 nm blue laser BD FACSCanto II Flow Cytometer Reference Manual...
  • Page 137 Figure B-6 5-3 configuration trigon detector array 633 nm red laser Appendix B: Optics Configurations...
  • Page 138 BD FACSCanto II Flow Cytometer Reference Manual...

  • Page 139: Appendix C: Supplies And Replacement Parts

    Appendix C Supplies and Replacement Parts To order supplies and options, contact your local BD Biosciences representative. For updated information and part numbers, refer to our website, bdbiosciences.com. • Instrument Supplies on page 140 • Consumables on page 142...

  • Page 140: Instrument Supplies

    Vented cap for waste tank 338922 Waste cap baffle 338505 Fluidics cart fuses: 2.5-A 250V Slo-blo Type T 343565 Loader front door assembly (Loader option only) 34401007 Loader side door assembly (Loader option only) 34349207 BD FACSCanto II Flow Cytometer Reference Manual...

  • Page 141: Other Replacement Parts

    Other Replacement Parts Item Part No. Sheath sensor probe (2-level) 338979 Waste sensor probe (2-level) 338978 Auxiliary sensor probe (1-level) 343835 Air filter (side door) 336303 Bal seal retainer 640116 Carousel kit, carousels 1–4 332727 Carousel kit, carousels 5–8 332728 Carousel kit, carousels 9–12 332729 Carousel kit, carousels 13–16...

  • Page 142: Consumables

    Monoclonal antibodies BD Biosciences BD FACS™ lysing solution BD Biosciences 349202 a. Refer to the BD Biosciences Immunocytometry Products Catalog or the BD Biosciences website (bdbiosciences.com). b. US Patent Nos. 4,654,312; 4,902,613; 5,098,849 BD FACSCanto II Flow Cytometer Reference Manual...

  • Page 143: Labware

    Labware Item Supplier Catalog No. 5-mL polystyrene test tubes, BD Biosciences 12 x 75-mm (BD Falcon tubes) (877) 232-8995 • uncapped, 125 per bag • 352052 • capped, 125 per bag • 352054 • capped, 25 per bag • 352058 •...
  • Page 144 BD FACSCanto II Flow Cytometer Reference Manual...

  • Page 145: Appendix D: Technical Specifications

    Appendix D Technical Specifications • Cytometer Specifications on page 146 • Fluidics Cart Specifications on page 150 • BD FACS Loader Specifications on page 151 For barcode reader specifications, refer to the information supplied by the manufacturer.

  • Page 146: Cytometer Specifications

    ≤146 kg (320 lb)—cytometer only, excluding Weight Loader and computer Maximum 168 kg (370 lb)—including Loader Power requirements 100/115/230 VAC (50–60 Hz) Current: 5A at 115 VAC 2.5A at 230 VAC Power consumption 500 W BD FACSCanto II Flow Cytometer Reference Manual...

  • Page 147: Environment

    Forward and side scatter sensitivity Platelets can be resolved from noise Forward scatter sensitivity 1 micron Side scatter sensitivity 0.5 micron Optics Laser Specifications The following Class 3B lasers are mounted on the BD FACSCanto II instrument. Wavelength Power Manufacturer Model (nm) (mW) Coherent...
  • Page 148 These lasers are contained within the instrument, therefore the BD FACSCanto II flow cytometer is a Class I (1) laser product. Excitation Optics Optical platform Fixed optical assembly Beam geometry (all lasers) 9 µm x 65 µm elliptical beam Emission Optics Collection lens Optical gel–coupled to flow cell...

  • Page 149: Fluidics

    Sheath consumption 1.1 L/hr, normal operation <1.0 mL/hr, standby Sample flow rates Assay dependent, controlled automatically by BD FACSCanto clinical software. Nominal rates: Low = 10 µL/min Medium = 60 µL/min High = 120 µL/min Sample acquisition rate 10,000 events/sec with <10% abort rate...

  • Page 150: Fluidics Cart Specifications

    Weight ≤82 kg (180 lb)—with tanks full Facilities No air supply or vacuum required Capacity BD FACSFlow cubitainer 20 L BD FACSClean solution cubitainer BD FACS shutdown solution cubitainer Waste tank 10 L BD FACSCanto II Flow Cytometer Reference Manual...

  • Page 151: Bd Facs Loader Specifications

    Accommodates up to 40 uncapped 12 x 75-mm tubes • BD Falcon polystyrene test tubes • BD Trucount tubes • BD FACS 7-color setup bead tubes • Thickness of accumulated labels ≤0.125 mm (5 mils) no more than 3 labels thick ≤1.07 mL...
  • Page 152 BD FACSCanto II Flow Cytometer Reference Manual...

  • Page 153: Index

    48 installing 46 symbologies 49 BD FACS Loader 29 achromatic focusing lens 20 BD FACS Sample Prep Assistant II 13 air filter, replacing 60 BD FACS shutdown solution air flow direction 62 cubitainer 27 air lock, removing 72...
  • Page 154 123 5-3 19, 134 emitted light 18 connecting power cords 24 ethanol 75 connection excitation optics 19 fluid port 27 level-sensor connection 27 container, waste 27 fiber optic cables 20 fiber optics 20 BD FACSCanto II Flow Cytometer Reference Manual...
  • Page 155 filters fluidics cart air, replacing 60 components 26 bandpass 113 controls 28 bubble 78 dimensions 150 changing 63 fuses, replacing 92 fluidic 26 maintenance 54, 68 longpass 22, 112 reconnecting tubing 86 optical theory 112 troubleshooting 98, 101 purging 57 fluorescence 111 removing an air lock 72 detection 148...
  • Page 156 24 scheduled 52 red 19 unscheduled 66 safety interlock 20 manual mode 40 solid state 20 mirror, longpass 22 specifications 147 modes troubleshooting 99 automatic loading 41 violet 19 manual loading 40 BD FACSCanto II Flow Cytometer Reference Manual...
  • Page 157 pressure, sheath fluid 107 priming, fluid lines 71 obscuration bar 19, 22 prisms 20 octagon 114 beam-shaping 20 optics 21, 23 pulse 120 operating temperature 147 area 122 optical sensors 34 height 122 Opticon LG2 Imager 30, 46 purging filters 57 optics 4-2 configuration 19 4-2-2 configuration 19...
  • Page 158 21, 23 troubleshooting 102 tube eject cylinder 17 guide 34, 37 about 17 sensor 17 wet 18 tubes compatible 35 lifter 34 sample volume 151 tubing about 17 network 16 unscheduled maintenance 66 BD FACSCanto II Flow Cytometer Reference Manual...

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