Page 1 BD FACSAria User’s Guide bdbiosciences.com Part No. 643632 Rev. A December 2007 BD Biosciences Asia Pacific Brazil Canada Tel (65) 6-861-0633 Tel (55) 11-5185-9995 Toll Free (888) 259-0187 San Jose, CA 95131-1807 Fax (65) 6-860-1593 Fax (55) 11-5185-9895 Tel (905) 542-8028...
Page 2 Becton, Dickinson and Company. 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: Table Of Contents
Limitations ..........Chapter 1: BD FACSAria Cytometer Components Fluidics Cart .
Page 4 Conflict Resolution During Sorting ......Chapter 3: Using BD FACSDiva Software Workspace Components ........
Page 5 Overview of Cytometer Setup and Tracking ..... . Cytometer Quality Control Using BD FACSDiva Software ... . .
Page 6 Unscheduled Maintenance ........BD FACSAria User’s Guide...
Page 7 Changing the Nozzle ........Adjusting the Nozzle Orifice .
Page 8 Creating Application Settings ....... . . Index viii BD FACSAria User’s Guide...
Page 9: About This Guide
For detailed information on software features, refer to the BD FACSDiva Software Reference Manual. The BD FACSAria User’s Guide 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
Print” means to choose Print from the File menu. Ctrl-X When used with key names, a dash means to press two keys simultaneously. For example, Ctrl-P means to hold down the Control key while pressing the letter p. BD FACSAria User’s Guide...
Page 11: Technical Assistance
• details of recent system performance For instrument support from within the US, call (877) 232-8995. For support from within Canada, call (888) 259-0187. Customers outside the US and Canada, contact your local BD representative or distributor. About This Guide...
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Page 13: Safety And Limitations
Safety and Limitations The BD FACSAria flow cytometer is equipped with safety features for your protection. Operate only as directed in the user’s guide. Do not perform instrument maintenance or service except as specifically stated. Keep this safety information available for reference.
Page 14: Laser Product Classification
The higher the classification number, the greater the potential hazard. The BD FACSAria flow cytometer is a Class I (1) laser product per 21 CFR Subchapter J and IEC/EN 60825-1:1994 + A2:2001. The lasers and the laser energy are fully contained within the instrument structure and call for no special work area safety requirements except during service procedures.
Page 15: Electrical Safety
Connect the equipment only to an approved power source. Do not use extension cords. If cords, plugs, or cables are damaged, immediately contact BD Biosciences for a replacement. • Do not remove the grounding prong from the power plug. Have a qualified electrician replace any ungrounded receptacles with properly grounded receptacles in accordance with the local electrical code.
Page 16: Biological Safety
To keep the cap dry, place it on the bench label- trap side up when it is not on the tank. If you see liquid in the waste cap trap, remove the drain plug and fully drain the liquid before you replace the plug. drain plug BD FACSAria User’s Guide...
Page 17: General Safety
Cell sorters that use droplet generation methods, like the BD FACSAria, can produce aerosols around the sample stream. When acquiring biohazardous samples, follow universal precautions at all times. Keep the sort block door closed during sorting. If you need to access the sort block, turn off the stream before opening the door.
Page 18: Precaution Labels
Precaution Labels The following precaution labels appear on the BD FACSAria flow cytometer or fluidics cart to indicate a potential hazard. Do not remove these labels. Use appropriate precautions to avoid injury by the indicated hazard. See the previous sections for more information.
Page 19 Label Location(s) Potential Hazard Ethanol tank cap Highly flammable material Ethanol On or near all removable Risk of exposure to covers and any place hazardous laser radiation where the laser beam can emerge from the CAUTION instrument VISIBLE AND/OR INVISIBLE CLASS 3B LASER RADIATION WHEN OPEN.
Page 20: Limitations
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 the buyer/ user's failure to install and maintain virus protection.
Page 21: Chapter 1: Bd Facsaria Cytometer Components
BD FACSAria flow cytometer enables multicolor analysis of up to 13 fluorescent markers and two scatter parameters at a time. The BD FACSAria system consists of three major components: a fluidics cart, a benchtop flow cytometer, and a workstation (see Figure 1-1 on page 22). Nearly all cytometer functions are operated from within BD FACSDiva software.
Page 22: Fluidics Cart
(although the cart can be hooked up to an in-house air source, if one is available). The air pumps provide pressure from 2 to 75 psi to accommodate a variety of cell sorting applications. Air pressure is adjusted within BD FACSDiva software.
Page 23: Containers And Connectors
The fluidics cart holds four 10-L containers (two sheath and two waste), and three 5-L auxiliary cleaning fluid containers (Figure 1-3). The cart can also accommodate up to two BD FACSFlow™ 20-L cubitainers in place of the four 10-L containers.
Page 24 Auxiliary Air Supply for 15 seconds every 8 hours (every 4 hours in an extremely humid climate). Doing this empties the cart condensation trap and prevents excess moisture from overflowing the trap or causing cart damage. BD FACSAria User’s Guide...
Page 25: Power And Operation
(Figure 1-5). A pressure reading of less than 80 psi or greater than 100 psi indicates that the fluidics cart is not functioning properly. If this occurs, contact your BD Biosciences service representative for assistance. Do not operate the cytometer outside the normal air pressure range.
Page 26: Flow Cytometer
Flow Cytometer The benchtop flow cytometer contains the major components for all three subsystems (fluidics, optics, and electronics). The BD FACSAria cytometer is relatively compact, with a much smaller footprint than most sorters with the same capabilities. The cytometer can be set up on a typical laboratory benchtop or table, and it requires only a 20-amp electrical outlet.
Page 27: Fluidics Components
Plenum Reservoirs on page 28 • Sample Injection Chamber on page 29 • Cuvette Flow Cell on page 30 • Nozzle on page 31 • Sort Block on page 32 • Sort Collection Chamber on page 35 Chapter 1: BD FACSAria Cytometer Components...
Page 28 Sheath fluid is pumped from the fluidics cart into a plenum reservoir inside the side door of the BD FACSAria flow cytometer (Figure 1-7). From there, the fluid travels to a second reservoir where it is held and pressurized until it flows into the cuvette flow cell.
Page 29 After a tube is loaded, the Load button changes to Unload. Click the Unload button to lower the loading port after data has been recorded. After each tube is unloaded, sheath fluid flushes the sample tubing inside and out to reduce Chapter 1: BD FACSAria Cytometer Components...
Page 30 Cuvette Flow Cell The cuvette flow cell is the heart of the BD FACSAria cytometer (Figure 1-9). Within the flow cell, hydrodynamic focusing forces particles through the cuvette in a single-file stream, where laser light intercepts the stream at the sample interrogation point.
Page 31 Nozzle The BD FACSAria cytometer is provided with two adjustable nozzles, 70 and 100 μm, that accommodate a variety of particle sizes. The nozzle is keyed to a fixed position at the end of the cuvette. Because the nozzle is below the interrogation point, optical alignment is not affected when the nozzle is changed.
Page 32 In this case, you can change the angle of the sort block by loosening the adjustment screws on either side of the deflection plates and rotating the sort block. Tighten the screws when the stream is re-centered in the aspirator. BD FACSAria User’s Guide...
Page 33 To avoid pinching your hands or fingers in the drawer, keep your hands away from the sort block during sorting. Figure 1-12 Aspirator drawer closed (left) vs open (right) Chapter 1: BD FACSAria Cytometer Components...
Page 34 During sample acquisition and sorting, the sort block door should be kept closed to help contain potential aerosols (Figure 1-13). Cell sorters that use droplet generation methods, like the BD FACSAria, can produce aerosols around the sample stream. Inhalation or contact with aerosols exposes you to biologically transmissible diseases.
Page 35 Two-way 15-mL collection tube holder An automated cell deposition unit (ACDU) that sorts into multiwell plates and onto microscope slides is available as an option. BD Biosciences also offers a temperature-control option to maintain the temperature of sorted samples during sorting.
Page 36: Optics System
Optics System The BD FACSAria cytometer uses innovative designs for both the excitation optics and collection optics. The optics can be viewed by opening the optics access door and the flow cell access door. See the following sections for more information.
Page 37 See Precaution Labels on page xviii for the placement of laser warning labels. Figure 1-15 Excitation optics pathway (behind laser shielding) prisms fiber optics prisms focusing lens cuvette flow cell upper camera Chapter 1: BD FACSAria Cytometer Components...
Page 38 Bandpass filters in front of each PMT allow fine-tuning of the spectral wavelengths that need to be collected. Since reflection is more efficient than transmittance, this design greatly increases the multicolor detection capabilities of the cytometer. BD FACSAria User’s Guide...
Page 39 At installation, the octagon and trigon arrays are set up with the filter and mirror combinations shown in Table 1-1 on page 40. You can rearrange these configurations according to the type of fluorochromes in your experiment. See Application Options on page 250 for more information. Chapter 1: BD FACSAria Cytometer Components...
Page 40 Trigon 530/30 Alexa Fluor® 430 (405-nm violet ™ — 450/40 Cascade Blue®, Pacific Blue laser) DAPI, Hoechst, Alexa Fluor® 405 a. The optical holder for the 488/10 BP filter also includes a 1.0 ND filter. BD FACSAria User’s Guide...
Page 41 • The lower camera generates an image used for the BD FACS™ Accudrop option. It enhances the ability to see side streams and assists in setting an accurate drop delay value.
Page 42: Cytometer Electronics
The cytometer circuit breaker is located next to the power cord (Figure 1-18). The switch will need to be reset if there is a power surge in the laboratory. BD FACSAria User’s Guide...
Page 43 Do not reset the button until the message appears. To do so, turn the button clockwise until the light turns off and the button returns to its original position. NOTE The emergency stop button does not turn off the lasers or shut down the cytometer main power. Chapter 1: BD FACSAria Cytometer Components...
Page 44: Workstation
Workstation Data acquisition and analysis, as well as most BD FACSAria cytometer functions, are controlled by BD FACSDiva software on a third-party PC workstation. The workstation includes a desktop computer, one or two monitors, and a color printer, and is equipped with the following applications: •...
Page 45: Chapter 2: Theory Of Operation
This chapter describes how the BD FACSAria cytometer works and how BD FACSDiva software components are used to operate different system components. For a general overview of the software, see Chapter 3. See the following sections for a description of these BD FACSAria functions: • Fluid Movement on page 46 •...
Page 46: Fluid Movement
(Figure 2-1). The following sections describe the controls used to move fluids through the BD FACSAria fluidics system. Figure 2-1 Fluidic movement via the fluidics system...
Page 47 Figure 2-2 Sheath pressure level After fluidics startup, sheath flow is controlled using the Stream button in the Breakoff window, which is labelled with the chosen sort pressure (Figure 2-3). When clicked, the button changes from a red “X” to a green checkmark, and sheath flows through the cuvette flow cell at the rate that is specified in the Sheath Pressure window.
Page 48: Sample Flow
10–120 μL/min. Figure 2-4 Load button and Flow Rate field in Acquisition Dashboard Note that the relatively longer sample tubing on the BD FACSAria cytometer results in a different flow rate between cells and beads. Thus, absolute counting using BD Trucount™ beads can give erroneous results.
Page 49 Hydrodynamic Focusing In the flow cell, pressurized sheath fluid surrounds the sample fluid to hydrodynamically focus the core stream of suspended cells into the center of the cuvette, where the particles are intercepted by the laser beam. The difference in pressure between the sheath fluid and the sample fluid can be used to vary the diameter of the sample core.
Page 50: Signal Generation
(SSC). FSC and SSC are related to certain physical properties of cells: • 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 side scatter forward scatter light source BD FACSAria User’s Guide...
Page 51: Fluorescent Signals
Fluorescent Signals 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 52: Signal Detection
Detector Arrays There are two types of detector arrays in the BD FACSAria flow cytometer: an octagon and trigons. The octagon detects SSC and up to seven fluorescent signals excited by the 488-nm (blue) laser. The trigons detect fluorescent signals excited by the 633-nm (red) and 405-nm (violet) lasers, respectively.
Page 53: Filters
Filters Optical filters modify the spectral distribution of light scatter and fluorescence directed to the detectors. Three kinds of filters are used in the detector arrays: longpass (LP) filters are used to steer light between the detectors within a detector array, while bandpass (BP) and neutral density (ND) filters allow fine-tuning of the spectral wavelengths that need to be collected (Figure 2-7).
Page 54 655 nm and 735 nm are detected at PMT-B; all wavelengths <655 nm are reflected to PMT-C, and so on. For a list of the longpass filters used in the detector arrays, see Table 1-1 on page 40. BD FACSAria User’s Guide...
Page 55 Bandpass Filters Bandpass (BP) filters transmit 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 56 For optimal detection of fluorescent light, a bandpass filter must always be installed in front of each detector. For a list of the bandpass filters used in the detector arrays, see Table 1-1 on page 40. BD FACSAria User’s Guide...
Page 57 100% The ND filters on the BD FACSAria allow approximately 10% of the light to be transmitted. You can find them in front of the FSC and SSC detectors. For applications involving small particles (eg, bacteria or platelets), you might need to remove the FSC ND filter.
Page 58: Detectors
Detectors within each detector array convert light signals into electrical signals that can be processed by the electronics system. There are two types of signal detectors in the BD FACSAria flow cytometer: the photodiode and photomultiplier tubes (PMTs). The photodiode is less sensitive to light signals than the PMTs, thus is used to detect the stronger FSC signal.
Page 59: Electronic Processing
Electronic Processing As cells or other particles pass through the focused laser beams, they scatter the laser light and can emit fluorescence. Because each laser beam is focused on a small spot and particles move rapidly through the flow cell, the scatter or fluorescence emission has a very brief duration—only a few microseconds.
Page 60: Pulse Parameters
Log checkbox to convert the display to a log scale. The Experiment Inspector contains an option to display log data on a four- or five-decade scale. (Refer to the BD FACSDiva Software Reference Manual for more information.) BD FACSAria User’s Guide...
Page 61: Laser Delay
The delay factor in BD FACSDiva software is used to realign the signals so they can be measured and displayed on the same time scale. Note that signals are aligned with respect to the blue laser, so the red laser signals always have a negative delay value.
Page 62: Sorting
Droplets detach from the stream a few millimeters downstream from the nozzle. The time between when a particle intercepts the laser and when it reaches the droplet breakoff point is determined using BD FACS Accudrop technology (see Drop Delay Overview on page 69).
Page 63: Drop Formation
Drop Formation The BD FACSAria cytometer is unique in that drop drive energy is constantly applied to the stream; droplets form as soon as you turn on the stream. Sample interrogation takes place upstream of the stream vibration so that analysis is not affected by the drop drive.
Page 64 Sweet Spot, the stream is shut off and sorting is stopped: the deflection plates shut off, the aspirator drawer closes, and the sample tube is unloaded. BD FACSAria User’s Guide...
Page 65 Pre-programmed values can be downloaded to the Breakoff window by selecting one of the nozzle sizes (70, 85, 100, 130 micron) from the Sort > Sort Setup menu. Note that changes to values in the Sort Setup windows (Breakoff and Side Stream) are automatically saved.
Page 66 Frequency field In general, the drop drive frequency should not need adjustment. (continued) BD Biosciences recommends using the default values that are entered with each Sort Setup mode. Drop 1 field The distance between the top of the image and the center of the first broken-off drop, from 100–600 pixels.
Page 67: Side Stream Formation
Side Stream Formation Side streams are formed when the voltage is on and you are sorting, or when you click Voltage, then Test Sort in the Side Stream window. The Side Stream window displays an image of the side streams as transmitted by the lower camera.
Page 68 Sets the amount of time between when an event is measured and the breakoff point, from 10–140 drops. The drop delay value determines which drop will be deflected. The drop delay value is set experimentally using BD FACS Accudrop technology. Auto Delay Opens the Auto Drop delay dialog.
Page 69 In general, the Phase never needs adjusting; you can keep the default value of zero. Drop Delay Overview The BD FACSAria cytometer includes integrated Accudrop technology to assist in setting an accurate drop delay value. Accudrop components consist of the following: •...
Page 70: Drop Charging
During sorting, when an event is identified within one of the sort gates, the drop containing the particle of interest is charged via the stream-charging wire in the nozzle. stream-charging wire BD FACSAria User’s Guide...
Page 71: Conflict Resolution During Sorting
BD FACSDiva software accurately measures particle position to within 1/32 of a drop. Mask settings determine how drops are deflected when sorting conflicts occur.
Page 72 Yield Masks between 0–32 will sort either one or two drops. Yield Masks cannot be used in conjunction with Phase Masks. Therefore, when the Yield Mask is greater than zero, the Phase Mask automatically reverts to zero. BD FACSAria User’s Guide...
Page 73 Purity Mask The Purity Mask setting defines how close, in 1/32-drop increments, a contaminating drop can be located before ignoring the drop being interrogated. For example, when the Purity Mask is set to 16, the drop being interrogated will not be sorted if a non-target particle falls within the first or last 8/32 of the leading or trailing drop.
Page 74 (Figure 2-22). Figure 2-22 Sorted drop with Phase Mask of 8 trailing drop (drop sorted) leading drop Phase Mask BD recommends using a Phase Mask of at least 8 when sorting single cells. BD FACSAria User’s Guide...
Page 75 Phase Masks cannot be used in conjunction with Yield Masks. Therefore, when the Phase Mask is greater than zero, the Yield Mask automatically reverts to zero. Sort Precision Modes Mask values can be combined in many different ways. By default, six Sort Precision modes are already defined—Purity, 4-Way Purity, Yield, Single Cell, Initial, and Fine Tune.
Page 76 In Fine Tune mode, all masks are set to zero to deflect the maximum number of drops. This mode is used to fine-tune the drop delay value. See Determining the Drop Delay on page 158 for more information. BD FACSAria User’s Guide...
Page 77 Defining New Precision Modes Default Precision modes cannot be edited or deleted. However, you can create new modes and then choose them from the Precision Mode drop-down menu. Select Sort > Sort Precision and click Add. The current sort mode is duplicated and the Mask fields are enabled. (Optional) Change the name of the mode in the Precision Mode field.
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Page 79: Chapter 3: Using Bd Facsdiva Software
All BD FACSAria cytometer functions are controlled using BD FACSDiva software. This chapter provides a general overview of the workspace components and describes software controls that are unique to the BD FACSAria cytometer. For an in-depth description of software components not described in this chapter, refer to the BD FACSDiva Software Reference Manual.
Page 80: Workspace Components
Getting Started with BD FACSDiva Software. When running BD FACSDiva with the BD FACSAria, two additional windows can be displayed by clicking the Sorting button on the Workspace toolbar. See Sorting Controls on page 92 for a description.
Page 81: Cytometer Controls
Acquisition Dashboard. See Acquisition Controls on page 91. Fluidics Controls Fluidics control of the BD FACSAria cytometer is completely automated by BD FACSDiva software. The software contains pre-programmed fluidics protocols that are activated by choosing the corresponding menu command from the Cytometer menu.
Page 82 Shutdown on page 178 for more details. Cleaning Modes BD FACSDiva software contains pre-programmed cleaning modes that are activated by choosing the corresponding menu command from the Cytometer > Cleaning Modes menu. See Internal Cleaning on page 181 for more information.
Page 83 Use the Sample Temperature command to set the temperature inside the sample injection chamber. You can select from one of the specified values, or select Off to turn off temperature control (Figure 3-2 on page 84). Chapter 3: Using BD FACSDiva Software...
Page 84: Fluidics Level Indicators
Fluidics Level Indicators BD FACSDiva software provides fluidics level indicators in the Cytometer window (Figure 3-3 on page 85). The sheath and waste indicators provide an approximate indication of the fluid levels in each tank. The DI, bleach, and ethanol tank indicators appear full until the fluid level descends below 20% of the tank capacity.
Page 85 Purge the filter as described in Purging the Bubble Filter on page 214. If you are unable to remove all of the air, replace the bubble filter as described in Changing the Bubble Filter on page 203. Chapter 3: Using BD FACSDiva Software...
Page 86: Cytometer Configuration
Application Guide for complete information on using CS&T. • Performance Tracking (LJ)—Opens the Performance Tracking feature within the main CS&T window. See the Cytometer Setup and Tracking Application Guide for complete information on using this feature. BD FACSAria User’s Guide...
Page 87 Cytometer Configuration Window The BD FACSAria cytometer is equipped with a specific set of lasers, filters, and dichroic mirrors. The Cytometer Configuration window lets you define which fluorochromes or cell parameters will be measured at each photomultiplier tube (PMT) detector. Within this window, you can define parameters for an unlimited number of fluorochromes, up to three lasers, and up to 15 detectors.
Page 88 Defining a Custom Configuration on page 252. For accurate data results, the cytometer optics must match the current cytometer configuration. BD FACSAria User’s Guide...
Page 89: Cytometer Status Report
See Figure 3-6 on page 90 for a sample report. For a full description of the Cytometer Status Report, refer to the BD FACSDiva Software Reference Manual. A BD FACSAria cytometer report includes the following additional information.
Page 90 Voltage and Voltage Center values. If the Sweet Spot is off, Breakoff and Gap values are shown. If the Sweet Spot is on, Drop 1 and target Gap values are shown. Figure 3-6 Cytometer Status Report BD FACSAria User’s Guide...
Page 91: Acquisition Controls
Acquisition Controls Along with the controls described in the BD FACSDiva Software Reference Manual, the following acquisition controls are available for the BD FACSAria cytometer. • Load—Lifts a tube into the sample injection chamber, starts sample agitation (if agitation is turned on), and starts acquisition of the sample.
Page 92: Sorting Controls
Sorting Controls All sorting on the BD FACSAria cytometer is controlled by BD FACSDiva software. Sorting controls are shown in Figure 3-7. Figure 3-7 BD FACSDiva sorting controls...
Page 93: Sort Menu
Sort Layout button on the Browser toolbar performs the same function.) • Open Sort Layout—Opens an existing sort layout. A sort layout must be selected in the Browser for this menu command to be enabled. Alternatively, double-click any sort layout to open it. Chapter 3: Using BD FACSDiva Software...
Page 94: Sort Setup
If you make changes to any of the settings, the changes are automatically saved when you exit BD FACSDiva software or when you switch to a different sort setup mode. When you restart, the most recently used set of values is restored.
Page 95 Drop Delay 47.00 30.00 27.00 16.00 Far left voltage Left voltage Right voltage Far right voltage Plate voltage 4,500 4,000 2,500 2,000 2nd Drop 3rd Drop 4th Drop Laser Delay (blue) 0.00 0.00 0.00 0.00 Chapter 3: Using BD FACSDiva Software...
Page 96: Sort Layout
2.00 2.00 2.00 4.00 a. Contact your BD Sales representative for information on the availability of the 85 and 130 micron nozzles. Sort Layout The Sort Layout window contains all sorting instructions and controls. The sort layout designates which device will be used to collect sorted particles and which particles will be sorted into each sort location.
Page 97 Figure 3-9 Sort layout for a frosted slide sort location field for a spot on a slide Chapter 3: Using BD FACSDiva Software...
Page 98 Choose Save Sort Reports option from drop-down menu. There are three choices for saving the sort report: • Save None—Sort Reports are not saved. • Save All—Automatically saves sort report each time the sort is stopped. BD FACSAria User’s Guide...
Page 99 Specify whether to save sort conflicts by selecting the Save Conflicts checkbox. This checkbox is enabled only when using a two- or four-tube layout. When selected, all sort conflicts are sorted into a default location. Chapter 3: Using BD FACSDiva Software...
Page 100 To clear all populations from a field, select the field, then select Clear All. Using Sorting Controls Sorting controls appear at the bottom of the Sort Layout window. Use these controls for the following functions. Access Stage Move Drawer Pause/Resume BD FACSAria User’s Guide...
Page 101 To display fewer counters in the Sort Layout window, click the View Counters button and select a menu option. The corresponding counter is hidden. (Only counters with a checkmark next to the name are displayed.) Chapter 3: Using BD FACSDiva Software...
Page 102 (Figure 3-11). When a target number is specified, the field displays the actual number of events along with the number of target events. A progress bar appears behind the Sort Rate counter field showing the progress of the sort. Figure 3-11 Sort Layout during sorting BD FACSAria User’s Guide...
Page 103: Sort Report
Acquisition Counters—threshold count, processed events count, electronic conflicts count, and elapsed time • Sort Counters—counter values per sort destination, or total sort count if sorting sequentially • Sort Layout—population(s), sort count, and target event count for each sort location field Chapter 3: Using BD FACSDiva Software...
Page 104 Figure 3-13 Typical Sort Report The Sort Report window contains a File menu where you can choose to print or export the report. Exported comma-separated value (CSV) files can be opened with a spreadsheet application such as Microsoft Excel®. BD FACSAria User’s Guide...
Page 105: Templates
Templates When you install BD FACSDiva software for the BD FACSAria cytometer, the following additional experiment templates are installed in the BD Export\ Templates directory: • Accudrop Drop Delay template—Contains a single specimen and tube, a gated plot on a standard worksheet, and a predefined sort layout. This experiment is used for setting the drop delay during sorting as described in Determining the Drop Delay on page 158.
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Page 107: Chapter 4: Running Samples
Cytometer Startup on page 108 • Checking Performance with Cytometer Setup and Tracking on page 117 • Cytometer Quality Control Using BD FACSDiva Software on page 118 • Data Collection on page 134 • Data Recording and Analysis on page 141 •...
Page 108: Cytometer Startup
Cytometer Startup Follow these steps to start up your BD FACSAria cytometer. Start up the workstation. Open the flow cell access door. The door is equipped with a laser interlock that blocks laser light when the door is open. Leave the flow cell access door open until you turn on the stream.
Page 109 See Sample Temperature on page 83. Start BD FACSDiva software, and log in with your user name and password. To start the software, double-click the application shortcut on the desktop.
Page 110: Setting Up The Breakoff
Do not exceed 70 volts. If you cannot achieve a drop breakoff at <70 volts, do the following: • Check the flow cell for air bubbles. If you see bubbles, turn the stream off and back on. BD FACSAria User’s Guide...
Page 111 • Make sure the sheath pressure and drop drive frequency are appropriate. If the amplitude is <10 volts, turn on Attenuation in the Side Stream window. Verify that the small satellite droplets are Good Poor merging with the large droplets. Satellite-merging is largely dependent on nozzle position.
Page 112 Check the fluid levels in the sheath and waste containers every time you use the cytometer. This ensures that you will not run out of sheath fluid during an experiment and that the waste container will not overflow. Fluidics level indicators are shown in the Cytometer window in BD FACSDiva software. waste bleach...
Page 113: Setting Up The Fluidics Cart
Refilling Containers You can refill a container directly on the fluidics cart without detaching any lines, or you can remove the container for refilling. Note that during operation, you can add fluid to a container through the large cap without any interruption to your experiment, but if you detach any lines, you will need to prime the system.
Page 114 Emptying the Waste Empty the waste tank daily and when the fluid indicator shows the waste is getting full. To prolong the life of the tank, BD Biosciences recommends that you switch to the alternate tank each time the waste is emptied.
Page 115 waste port disposable cap trap waste tank Disconnect the waste container’s sensor and fluid line connectors from their respective ports on the fluidics cart. fluid line sensor The waste tank can become pressurized when the cytometer is running. Always disconnect the tank from the wet cart before you empty it. Wait at least 1 minute for pressure to dissipate before you open the container.
Page 116 If one month has passed since you last changed the cap, replace the filter cap with a new one. When you replace the cap, write the date on the new cap as a reminder. Waste (A) space for date 338677 Rev A BD FACSAria User’s Guide...
Page 117: Checking Performance With Cytometer Setup And Tracking
Cytometer Setup and BD FACSDiva software Tracking With the release of BD FACSDiva software 6.0 and later, there are two options for ensuring consistent performance from the cytometer. Using the Cytometer Setup and Tracking (CS&T) module. See the overview below and refer to the Cytometer Setup and Tracking Application Guide for complete information on using CS&T.
Page 118: Checking Performance With Cytometer Setup And Tracking
For examples of fluorescent particles that can be used for cytometer QC, see Cytometer Setup Particles on page 246. NOTE QC results are affected by laser and fluidics performance. BD strongly recommends following the laser and fluidics maintenance procedures in Chapter 6.
Page 119: Cytometer Quality Control Using Bd Facsdiva Software
NOTE When you select Cytometer > View Configurations, the cytometer disconnects from the BD FACSDiva interface and connects to the CS&T module. While the CS&T module is running, BD FACSDiva is in a holding mode and does not accept any user input. When CS&T is closed, BD FACSDiva software becomes active again.
Page 120: Setting Up Cytometer Configuration
Close the CS&T module by selecting File > Exit. The BD FACSDiva interface is now active and accepting user input. See Figure 4-4 and Figure 4-5 for error messages that can occur if the sort setup or sheath pressure are different between the cytometer configuration in CS&T and the values in BD FACSDiva software.
Page 121 This section describes how to optimize area scaling and laser delay using the QC template that is included with BD FACSDiva software. Setting Up the QC Experiment Follow the steps in this section to set up an experiment for instrument QC.
Page 122: Preparing Qc Particles
Browser toolbar. Rename the folder Cytometer QC or create a Cytometer QC folder inside another folder. Refer to the BD FACSDiva Software Reference Manual for ideas on how to organize experiments. To place an experiment inside a folder, select the folder before you create the experiment.
Page 123 Adjusting Area Scaling for the Blue Laser Open the QC experiment by double-clicking the experiment icon. Verify that the Area Scaling worksheet is displayed in the Worksheet window. Click to set the current tube pointer next to the tube with today’s date in the Browser.
Page 124 10. Adjust the FSC area scaling until the FSC-A signal matches the FSC-H signal, if needed. Click the Laser tab in the Cytometer window and check at the FSC Area Scaling value. BD FACSAria User’s Guide...
Page 125 • If the FSC-A signal is lower than FSC-H, increase FSC area scaling. • If the FSC-A signal is higher than FSC-H, decrease FSC area scaling. Figure 4-7 FSC area scaling factor set incorrectly (left) and correctly (right) Chapter 4: Running Samples...
Page 126 Click the Laser tab in the Cytometer window. Adjust the area scaling factor for the blue laser. • If the FITC-A signal is lower than FITC-H, increase area scaling. • If the FITC-A signal is higher than FITC-H, decrease area scaling. BD FACSAria User’s Guide...
Page 127 Adjusting Area Scaling and Laser Delay for the Red and Violet Lasers The laser delay factor in BD FACSDiva software is used to realign the signals so they can be measured and displayed on the same time scale. Signals are aligned...
Page 128 • If the APC-A signal stayed the same, then go to step e. • If the APC-A signal decreased, then follow steps c through e. Click in the Delay field for the red laser. Adjust the laser delay value until the maximum APC-A signal is achieved. BD FACSAria User’s Guide...
Page 129 Before Adjustment After Adjustment Reset the window extension to the appropriate setting (typically 2). Adjust the red laser area scaling until the APC-A signal matches the APC-H signal, if needed. Click the Laser tab in the Cytometer window. • Increase the area scaling factor if the APC-A signal is lower than APC-H.
Page 130 Once the tube has finished recording, the tube icon changes to a tube with a disk. Also, the tube will now have cytometer settings associated with it. tube with data tube without data After data has been recorded, click Unload in the Dashboard and remove the tube. BD FACSAria User’s Guide...
Page 131 (Optional) Select File > Print. Keep a record of the daily QC results for future reference. By comparing results from day to day, you will be able to monitor instrument performance. An example of recorded data is shown in Figure 4-9. An example QC log sheet is provided at the end of this chapter.
Page 132 QC particles. Highlight the last recorded data file in your QC experiment and find the area scaling values in the Tube tab. Type these numbers into the Cytometer window, Laser tab. BD FACSAria User’s Guide...
Page 133: Reusing The Qc Experiment
tube tab area scaling values Ensure that the red and violet laser delays are set appropriately by setting the window extension to 0 and verifying that the area signal is not lost. Adjust the laser delay, if necessary. Reset the window extension to the appropriate value before verifying the remaining area scaling factors.
Page 134: Tracking Qc Results
CS&T module. Compensation will be automatically calculated using the Compensation Setup feature. For more information about this feature, refer to the BD FACSDiva Software Reference Manual. If you are performing compensation manually, not all steps will apply.
Page 135: Data Collection
(Optional) Create a folder for your experiment. Select the icon for your database and press Ctrl-N; rename the folder appropriately. Refer to the BD FACSDiva Software Reference Manual for ideas on how to organize experiments. To place an experiment inside a folder, select the folder before you create the experiment.
Page 136: Setting Up The Workspace
Apply Application Settings. See Figure 4-10 on page 137. Refer to Appendix B for instructions on creating Application Settings. If you are not using CS&T, you must adjust area scaling and PMT voltages for your sample. BD FACSAria User’s Guide...
Page 137 Figure 4-10 Application Settings window Select the application setting for your sample and click Apply. See Figure 4-11 for a typical error message if there are any mismatches between the application and cytometer settings. Figure 4-11 Example mismatch error message Select Experiment >...
Page 138 (labels) that require different compensation values. This is especially noticeable in tandem conjugates due to lot-to-lot variation. Refer to the BD FACSDiva Software Reference Manual for more information about this feature. A compensation specimen is added to the experiment, along with a stained control tube for each compensation control.
Page 139: Calculating Compensation
Since the application settings have already been optimized for your sample, the cytometer settings should require little or no adjustment. Figure 4-12 Voltages adjusted Click the Threshold tab and adjust the FSC threshold, if needed. Set the threshold to remove most of the debris without cutting off the singlet population (Figure 4-12).
Page 140 To keep track of compensation setups, include the experiment name, date, or both in the setup name. NOTE BD Biosciences recommends that you always visually and statistically inspect automatically calculated overlap values. The means of the positive controls should be aligned with the means of the negative.
Page 141: Data Recording And Analysis
Once you have optimized the cytometer electronics for your sample type, you are ready to record and analyze data. Before you record data, BD Biosciences recommends that you gate out doublets in order to record only singlet events. The Doublet Discrimination Gating template provides gated plots for this purpose.
Page 142: Data Recording And Analysis
• Select Experiment > Experiment Layout. • On the Acquisition tab, select the events to record field for all specimen tubes, and select or enter 5,000 events. • Click OK. BD FACSAria User’s Guide...
Page 143: Setting Up The Experiment
Setting Up the Global Worksheet A global worksheet will be used to perform doublet discrimination, and to set up plots to preview and record data. Click the Worksheets View button ( ) on the Worksheet toolbar. The global worksheet is shown. If you are using the Doublet Discrimination Gating template, the worksheet shows the predefined plots and gates used to distinguish singlets from doublets.
Page 144: Setting Up The Global Worksheet
• Select the two plots. • In the Inspector, select the FSC Gate checkbox. Arrange the fluorescent plots so they fill the page vertically. For an example, see Figure 4-16 on page 148. BD FACSAria User’s Guide...
Page 145 Recording Data This section describes how to adjust the gates to eliminate doublets and record singlet events. Move the current tube pointer to the 4-color_001 tube. Install the first mixed sample tube onto the loading port and click Load. Change the Events to Display to 5,000 events. Adjust the Scatter Gate to encompass the singlet events.
Page 146: Recording Data
Create a rectangle gate to capture the PerCP-Cy5.5 beads. Rename each population in the population hierarchy. Press the Enter key twice to move to the next population without using the mouse. Right-click either fluorescent plot and select Create Statistics View. BD FACSAria User’s Guide...
Page 147: Analyzing Data
A statistics view is added to the worksheet. Right-click the statistics view and select Edit Statistics View. Edit the statistics view as follows: • In the Header tab, select the checkbox to Use 2 columns for display. • On the Populations tab, deselect the checkboxes for all populations except FITC, PE, PerCP-Cy5.5, and APC.
Page 148 (Optional) Print the analysis. Figure 4-16 Sample analysis for mixed-bead tube BD FACSAria User’s Guide...
Page 149 Performing Batch Analysis Batch analysis allows you to automatically advance through a selected set of tube data when using a global worksheet. Do the following to set up batch analysis: Verify that the global worksheet you will be using for analysis is displayed in the worksheet window.
Page 150: Performing Batch Analysis
When the process is finished, a message similar to Figure 4-17 displays. Figure 4-17 Batch analysis complete Sample QC Log A sample QC log is provided on the following page. This log can be photocopied or used as a guide in designing your own QC log. BD FACSAria User’s Guide...
Page 151: Sample Qc Log
Chapter 4: Running Samples...
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Page 153: Chapter 5: Sorting
Sorting You can program BD FACSDiva software to sort a specified number of particles from multiple populations into a variety of sorting devices, including tubes, plates, and slides. Collection devices are provided for sorting into 1-mL, 12 x 75-mm, and 15-mL tubes; hardware for sorting into plates and slides is available as an option.
Page 154: Setting Up For Sorting
Start up the cytometer, the computer, and perform cytometer QC. • For startup, see Cytometer Startup on page 108. • For cytometer QC, see Cytometer Quality Control Using BD FACSDiva Software on page 118. • If you are using the CS&T module for performance checking, see Checking Performance with Cytometer Setup and Tracking on page 117.
Page 155: Setting Up For Bulk Sorting
Use gating tools and subsetting methods to define the population(s) of interest. Examples of gating analysis can be found in Analyzing Data on page 146 and in Getting Started with BD FACSDiva Software. Define a sort layout for the tube containing the defined sort populations and proceed with sorting.
Page 156 See External Cleaning on page 180. Turn on Test Sort and optimize the side streams. See Figure 5-2. Adjust the voltage sliders to view the required number of streams. BD FACSAria User’s Guide...
Page 157 Figure 5-2 Turning on Test Sort Test Sort button Voltage sliders If you cannot see a stream image or the image is dim, adjust the micrometer dial on the diode laser to better view the streams (Figure 1-17 on page 41). Adjust the 2nd, 3rd, and 4th Drop settings to tighten the center stream and fine-tune the side streams, if needed.
Page 158: Determining The Drop Delay
Click the Voltage control ( ) to turn off the deflection plates. Determining the Drop Delay BD FACS Accudrop technology is used to determine the optimal drop delay setting for your sorting application. For more information, see Drop Delay Overview on page 69.
Page 159: Sorting Beads To Determine The Drop Delay
Expand Specimen_001 and Tube_001. Set the current tube pointer to Tube_001. Open the sort layout by double-clicking it. Sorting Beads to Determine the Drop Delay Load a tube filled with a suspension of Accudrop beads (approximately 2 drops of beads in 0.5 mL PBS). In the Laser tab of the Cytometer window, set the window extension to zero.
Page 160 There is no need to collect the beads. When the drawer is closed, the beads are sorted to waste. Adjust the micrometer dial (Figure 1-17 on page 41) to obtain the brightest bead spot on the center stream. Click the Optical Filter control in the Side Stream window. BD FACSAria User’s Guide...
Page 161 This control moves the emission filter that allows you to view the Accudrop beads in front of the lower camera. When the control is clicked, the image switches from a raw image to a processed (digitized) image. The two boxes indicate the region of the image where the left and center stream intensities are calculated during image processing;...
Page 162: Determining The Drop Delay - Automatic Method
Coarse passes are used to find initial drop delay within 2 drops of the ideal. The coarse passes are faster than fine-tune passes. Fine-tune passes are used to locate the drop delay value that is ideal under current conditions. BD FACSAria User’s Guide...
Page 163: Using Auto Drop Delay
Using Auto Drop Delay Set up an experiment for drop delay as described in Setting Up the Experiment on page 158. Load a tube filled with a suspension of Accudrop beads (approximately 2 drops of beads in 0.5 mL PBS). Adjust the flow rate to achieve these values of events per second: 70 micron = 1,000 to 3,000 85 micron = 800 to 2,000...
Page 164: Sorting
Sorting Before beginning the sort, do the following: • Perform the steps outlined in Setting Up for Sorting on page 154. • Use gating tools and subsetting methods to define the population(s) of interest. BD FACSAria User’s Guide...
Page 165: Setting Up The Experiment
Examples of gating analysis can be found in Analyzing Data on page 146 and in Getting Started with BD FACSDiva Software. NOTE Gates drawn on a biexponential scale can be used for sorting; however, the cytometer will sort on a log scale. Therefore, a gate that crosses the zero boundaries will sort all events below zero into that gated population.
Page 166: Starting And Monitoring The Sort
The deflection plates turn off automatically each time a tube is unloaded. If you do not turn them back on before beginning a sort, a dialog box appears where you can turn on the plates and open the aspirator drawer by clicking BD FACSAria User’s Guide...
Page 167 Verify that the current tube pointer is indicating the appropriate tube in the Browser, then click Sort. Click OK if you are prompted to open the aspirator drawer or turn on the deflection plates. If you click Cancel, sorting will begin with the deflection plates off and the drawer closed.
Page 168 To clear a clogged nozzle, see Cleaning the Nozzle on page 209. (Optional) You can print the Sort Report at this time, or open the report later and print it then. You can also export the report. BD FACSAria User’s Guide...
Page 169: Setting Up For Sorting Into A Plate Or Slide
Setting Up for Sorting Into a Plate or Slide The following sections describe how to set up for sorting into a plate or slide. For general guidelines, see Setting Up for Sorting on page 154. Installing the Sorting Hardware Any cytometer surface that comes in contact with biological specimens can transmit potentially fatal disease.
Page 170 When sorting onto a slide, sorting proceeds in rows across the short end of the slide, and in columns along the long end of the slide. Make sure that you set up your sort layout accordingly. See Figure 5-7 on page 171. BD FACSAria User’s Guide...
Page 171: Setting Up The Stream
Figure 5-7 Sort order on a slide (A3) (A2) (A1) Setting Up the Stream This section describes how to optimize side stream deflection and how to adjust the Home location. When sorting into a plate or onto a slide, the stage is pre-programmed to move a set distance between wells on a plate or spots on a slide.
Page 172 (Figure 1-17 on page 41) to better view the streams. Select Sort > Home Device. In the Device Setup dialog box, select the collection device you are using and click Go to Home. Test Sort button The stage moves to the pre-programmed Home position. BD FACSAria User’s Guide...
Page 173 Double-click the Test Sort button to deposit a drop at the Home location. Inspect the collection device to see where the drop was deposited. If you need to move the stage to the front, close the Device Setup dialog box and click the Access Stage control in the Sort Layout window. Wipe the collection device dry and place it back on the tray support.
Page 174: Creating A Custom Device
You can program the ACDU stage to sort into any grid configuration. Create a custom device by entering the number of rows and columns and setting the Home and Farthest locations. BD FACSDiva software calculates the increment between rows and columns to determine the sort locations. The Home and Farthest locations for a 96-well plate are A1 and H12, respectively.
Page 175 Select the text in the Name field and enter a new name. Enter the number of sort location Rows and Columns. A device can have up to 60 rows and 25 columns. Use the Arrow keys and the Test Sort button to set the Home location, then click Set Home.
Page 176 Select the name of the custom device to be deleted in the Custom Devices dialog box. Click Delete. The device is deleted from the Custom Device list, but is retained within any sort layouts where it was used. BD FACSAria User’s Guide...
Page 177: Chapter 6: Shutdown And Maintenance
Shutdown and Maintenance The BD FACSAria cytometer is designed to require minimum maintenance. However, to preserve the reliability of the cytometer, you must regularly perform basic preventive maintenance procedures. This chapter explains routine maintenance procedures you should follow to keep your cytometer in good condition.
Page 178: Daily Shutdown
See Setting Up the Fluidics Cart on page 112. If you disconnect the DI water tank for refilling, prime the system before continuing. The cytometer starts the fluidics shutdown cycle. This removes sheath from the lines and plenum and replaces it with DI water. BD FACSAria User’s Guide...
Page 179 When prompted, install a tube containing approximately 3 mL of cleaning fluid such as BD™ FACSRinse solution (BD Catalog No. ), then 340346 click OK. The cytometer loads the tube and cleans the cuvette with the cleaning solution. When prompted, install a tube containing approximately 3 mL of clean DI water, then click OK.
Page 180: External Cleaning
Turn off the cytometer main power. Quit BD FACSDiva software and shut down the computer. During shutdown, you will normally hear a hiss, which is caused by condensed water discharging from the fluidics cart pumps. Turn off power to the cytometer at least once a day. If your laboratory runs...
Page 181: Scheduled Maintenance
FSC vs SSC dot plot Internal Cleaning BD FACSDiva software includes five pre-programmed cleaning modes that can be used alone, or in combination to provide the required level of cleaning. The following sections describe the different cleaning modes. See Table 6-1 on page 182 for an overview of each mode.
Page 182 Some of the internal cleaning modes require that you turn off the stream. Remember to open the flow cell access door to block the lasers before you turn off the stream. Running the lasers without the stream can degrade the performance of the cuvette flow cell. BD FACSAria User’s Guide...
Page 183 Sample Line Backflush After a sample tube is unloaded, the sample line tubing within the sample injection chamber is automatically flushed inside and out with sheath fluid to eliminate potential sample carryover. Use the Sample Line Backflush command to perform additional backflushing of the inside of the sample line after a tube is unloaded.
Page 184 Clean Flow Cell Use the Clean Flow Cell command to run a tube of cleaning fluid such as BD FACSRinse solution through the sample line and flow cell, without sheath running. NOTE After the procedure is complete, the cleaning fluid remains in the sample line and flow cell until the stream is restarted.
Page 185 Prime After Tank Refill Use the Prime After Tank Refill command to prime the fluid lines if a fluidics container was disconnected for refilling. Open the flow cell access door and turn off the stream. Select Cytometer > Cleaning Modes > Prime After Tank Refill. Select the checkboxes for the tanks that were refilled, then click OK.
Page 186 Use the ethanol option to decontaminate the lines with ethanol. The remaining option is described in Clean Bulk Injection Chamber on page 191. Consult with your BD representative before running any other cleaning solutions. All biological specimens and materials coming into contact with them can transmit potentially fatal disease.
Page 187 • Reduce the sheath pressure to 10 psi. • Using a paper towel to cover the quick-disconnect coupling, disconnect the sheath line from the filter outlet tubing at the top of the filter. Figure 6-1 Disconnecting the sheath line above and below the filter sheath line (outlet) quick-disconnect couplings metal clip...
Page 188 Select Cytometer > Cleaning Modes > Long Clean. If you have not already turned off the stream, a message appears reminding you to do so. Select a Long Clean option and click OK. Select from the following: BD FACSAria User’s Guide...
Page 189 (Optional) Remove the plenum reservoirs and wipe them down with 10% bleach, followed by DI water. BD Biosciences recommends that you remove the plenum reservoirs only for the most stringent decontamination. You do not need to perform this step each time you run a Long Clean.
Page 190 Left and Right when you remove them. receiver reservoir Pull off the tubing connected to each reservoir. Wipe the reservoirs with 10% bleach, followed by DI water. After wiping, dry them with a clean, lint-free cloth. BD FACSAria User’s Guide...
Page 191 Clean the float and receiver for each plenum reservoir. Use a spray bottle to clean the floats. Use a cloth soaked with bleach or ethanol to wipe down the receivers. The floats are fragile. Handle them with care. Do not spray ethanol into the cytometer behind or below the plenum reservoirs.
Page 192 Air-dries the chamber and then opens it A message appears when cleaning is complete. Click OK to dismiss the completion message. Use a clean, dry cloth to remove residual ethanol from the loading port. Before using the system, run fluidics startup. BD FACSAria User’s Guide...
Page 193 Prepare for Aseptic Sort Use the Prepare for Aseptic Sort command when you want to decontaminate the entire sheath path with 70% ethanol or 10% bleach, from the fluid line starting at the sheath inlet port in the fluidics cart to the nozzle outlet. For the most complete decontamination, autoclave the sheath container and install a new sheath filter before sorting.
Page 194 This procedure requires approximately 250 mL of bleach or ethanol. Remove the sheath filter on the back of the fluidics cart by pressing the tabs on each quick-disconnect coupling. coupling coupling Install bypass tubing in place of the sheath filter. BD FACSAria User’s Guide...
Page 195 bypass tubing Detach the sheath tubing and the ethanol or bleach tubing from their respective ports on the fluidics cart. Figure 6-3 Sheath and ethanol ports ethanol tubing sheath tubing Plug the ethanol or bleach tank quick-disconnect into the sheath port. Cleaning the Lines with Ethanol or Bleach Install a tube containing ethanol or bleach onto the loading port.
Page 196 Do not run ethanol/bleach longer than is necessary to decontaminate. NOTE Click Cancel only if you want to cancel the cleaning mode. Do not click Cancel to stop running ethanol/bleach. The cytometer stops running the ethanol/bleach sample, unloads the tube, and stops the stream. BD FACSAria User’s Guide...
Page 197 Replacing the Ethanol or Bleach with Sheath Fluid Now that all fluid lines have been cleaned, the system prompts you to install an autoclaved sheath container on the fluidics cart. Follow these steps to complete the cleaning procedure. At the prompt, detach the ethanol or bleach line from the sheath port on the fluidics cart and connect it to the ethanol or bleach port (Figure 6-3 on page 195).
Page 198: Purging Filters
This ensures that the filters will not dry out. Unscrew the bleeder valve on the top of the filter. bleeder valve Wait until fluid seeps out through the valve. Close the valve. BD FACSAria User’s Guide...
Page 199: Changing Fluid Filters
Wipe up any excess fluid that might have dripped onto the fluidics cart. Changing Fluid Filters BD Biosciences recommends changing the fluid filters every 3–6 months, or when increased debris in an FSC vs SSC plot indicates that the sheath filter needs to be replaced.
Page 200: Changing The Sample Lines
HPLC valve and the cuvette flow cell needs changing only when it is kinked or clogged. To withstand the high pressures generated by the BD FACSAria flow cytometer, the sample lines are attached at each end using a two-piece compression fitting, where a cone-shaped ferrule is compressed onto the tubing as the connecting nut is tightened.
Page 201 Perform the following steps to replace the tubing. The same procedure can be used to replace either the primary or secondary sample line. Turn off the stream and lasers (if needed). Make sure the loading port is in the unload position. Unscrew the connecting nuts on each end of the tubing, and pull out the sample line.
Page 202 Turn on the stream, load a tube of water, and make sure none of the fittings are leaking. If needed, unload the tube, turn off the stream, and tighten the fittings. After tightening, if leaking still occurs, replace the ferrule. BD FACSAria User’s Guide...
Page 203: Changing The Air Filters
Changing the Air Filters The BD FACSAria cytometer has two air filters: one in the sort collection chamber door and one in the side door. •...
Page 204 Set the sheath pressure back to its original setting. In BD FACSDiva software, run fluidics startup. During fluidics startup, gently tap the bubble filter to dislodge air bubbles. Purge the bubble filter as described in Purging the Bubble Filter on page 214.
Page 205: Unscheduled Maintenance
Unscheduled Maintenance There are several cytometer components that should be cleaned periodically or checked for wear and replaced if necessary. See the indicated sections for the following maintenance procedures. Procedure Recommended Frequency Changing the Nozzle on this page As needed when running different size particles Adjusting the Nozzle Orifice on page 207 When satellite drops are not merging...
Page 206 NOTE After changing the nozzle, you might need to adjust the angle of the sort block to re-center the stream in the aspirator. To do so, loosen the adjustment screws on either side of the deflection plates and rotate the sort block (see BD FACSAria User’s Guide...
Page 207: Adjusting The Nozzle Orifice
Figure 1-11 on page 32). Tighten the screws when the stream is centered in the aspirator. For further assistance, see Troubleshooting the Stream on page 220. Adjusting the Nozzle Orifice Once a nozzle’s orifice is aligned to the cuvette, the nozzle should not need to be adjusted.
Page 208 (black) half of the cam, the nozzle adjustment is out of range. For proper nozzle operation, do not adjust the cam outside its adjustment range. Turn on the Sweet Spot when the drop pattern is stable. BD FACSAria User’s Guide...
Page 209: Cleaning The Nozzle
Cleaning the Nozzle Use one of the following methods to clean the nozzle when the stream appears blocked or distorted. To verify that the nozzle is clogged, examine the tip under a microscope. Figure 6-9 shows an example of an unclogged nozzle tip. Figure 6-9 Magnified view of a nozzle tip O-ring All biological specimens and materials coming into contact with them can...
Page 210 (see Figure 1-11 on page 32). Tighten the screws when the stream is centered in the aspirator. For further assistance, see Troubleshooting the Stream on page 220. BD FACSAria User’s Guide...
Page 211: Cleaning The Camera Windows
Cleaning the Camera Windows • Clean the lower camera window and the diode laser window when you have trouble viewing the side streams or you cannot set the drop delay using Accudrop. Wipe the windows with a soft, lint-free cloth soaked with DI water, and then dry the windows.
Page 212 Breakoff window when you intercept either opening. upper camera window Gently wipe the upper camera window, and then the strobe lens (opposite the window) to remove any saline. Repeat with spectral-grade methanol or absolute ethanol until clean. BD FACSAria User’s Guide...
Page 213: Cleaning The Optical Filters
Re-install the nozzle and deflection plates. See Changing the Nozzle on page 205 for details. Adjust the plates so there is a gap of approximately 0.28 in. (0.7 cm) between them at the top. Cleaning the Optical Filters Optical filters should be inspected occasionally and cleaned as necessary. The frequency will depend on how often the filters are handled.
Page 214: Purging The Bubble Filter
Change the pressure to 10 psi and click OK. Using a paper towel to cover the coupling, disconnect the sheath line from the filter outlet tubing by pressing the tab on the quick-disconnect coupling (Figure 6-10 on page 215). BD FACSAria User’s Guide...
Page 215 Figure 6-10 Filter outlet tubing connected to sheath line sheath line quick-disconnect coupling filter outlet tubing Connect the sheath line to the filter purge tubing (Figure 6-11). Figure 6-11 Filter purge tubing connected to sheath line sheath line filter purge tubing Turn on the stream.
Page 216 Purge the filter as described in Purging the Bubble Filter on page 214. If you are unable to remove all of the air, replace the bubble filter as described in Changing the Bubble Filter on page 203. BD FACSAria User’s Guide...
Page 217 Chapter 6: Shutdown and Maintenance...
Page 218 BD FACSAria User’s Guide...
Page 219: Chapter 7: Troubleshooting
Additional troubleshooting information can be found in the BD FACSDiva Software Reference Manual. If additional assistance is required, contact your local BD Biosciences technical support representative. See Technical Assistance on page xi. Troubleshooting suggestions in this chapter are grouped under the following headings: •...
Page 220: Troubleshooting The Stream
• If debris is visible, clean the nozzle. See Cleaning the Nozzle on page 209. • If the nozzle appears damaged, replace it. See Changing the Nozzle on page 205. BD FACSAria User’s Guide...
Page 221 Troubleshooting the Stream (continued) Observation Possible Causes Recommended Solutions No stream or dripping Nozzle inserted improperly Turn off the stream. Remove the stream nozzle. See Changing the Nozzle on page 205 for instructions. Clogged or damaged nozzle Turn off the stream, remove the nozzle, and examine the nozzle tip under a microscope.
Page 222 Unstable stream Debris in flow cell or nozzle Remove the nozzle and run the stream with no nozzle in place for approximately 10 seconds. (Click the Stream control on, then off.) Sonicate the nozzle and re-install BD FACSAria User’s Guide...
Page 223 Troubleshooting the Stream (continued) Observation Possible Causes Recommended Solutions Leaking or spraying Defective nozzle O-ring Replace the O-ring. around nozzle Nozzle inserted improperly Turn off the stream. Remove the nozzle. See Changing the Nozzle on page 205 for instructions. Extra O-ring is blocking the Remove the nozzle and use a nozzle cotton swab to clear out the...
Page 224: Troubleshooting The Breakoff
Turn off nozzle and nozzle, adjust nozzle, clean as described in attenuation in re-insert it. the opening, it, and then Cleaning the the Side and re-insert it. re-insert it. Camera Stream Windows on window. page 211. BD FACSAria User’s Guide...
Page 225: Sorting Troubleshooting
Sorting Troubleshooting Observation Possible Causes Recommended Solutions Unstable breakoff while Residual ethanol in system Allow the system to run until the Sweet Spot is engaged breakoff stabilizes. Target Drop 1 value is out of Use an actual Drop 1 value for the range for drop spacing target.
Page 226 Voltage sliders are set too far Move sliders in or out so they reversal, where the in or too far out. control the correct side streams. streams appear to be associated with the wrong voltage slider. BD FACSAria User’s Guide...
Page 227 Sorting Troubleshooting (continued) Observation Possible Causes Recommended Solutions No deflection or Insufficient voltage • Increase the plate voltage. insufficient deflection • Increase the side-stream voltages using the slider controls. Stream-charging wire is loose Verify the stream-charging wire is or missing inserted all the way into the barb.
Page 228 Viewing sort layout for Open or create a sort layout for another tube the current acquisition tube. Sort layout counters not Viewing sort layout for Open or create a sort layout for updating another tube the current acquisition tube. BD FACSAria User’s Guide...
Page 229 Sorting Troubleshooting (continued) Observation Possible Causes Recommended Solutions High sort conflict rate Event rate is too high for Decrease the event rate. drop drive frequency Gating conflict Verify the gating hierarchy. Purity mask is too high Decrease the purity mask. Erratic sort rate Flow rate is too high Decrease the flow rate.
Page 230: Acquisition Troubleshooting
Viewing plots for a different Double-click the current tube in tube the Browser to display the plots for that tube. Incorrect population(s) in Right-click the plot and choose plot Show Populations. Verify that the appropriate populations are displayed. BD FACSAria User’s Guide...
Page 231 Acquisition Troubleshooting (continued) Observation Possible Causes Recommended Solutions No events in plots after Uncolored events in plot • Format the plot to display all clicking Acquire Data events. (continued) • Assign a color to the population displayed in the plot. •...
Page 232 Right-click the plot and choose plot Show Populations. Verify that the appropriate populations are displayed. Incorrect drawing order Verify that the required population is not hidden by another population. Right-click the plot and choose Order Populations by Count. BD FACSAria User’s Guide...
Page 233 Possible Causes Recommended Solutions Erratic event rate Sample aggregates Filter the sample. Bulk injection O-ring is worn Contact your BD Biosciences service engineer. Sample is contaminated Re-stain the sample, making sure the tube is clean. Sheath container low Fill the sheath container.
Page 234 200. Sample aggregates Filter the sample. Memory is full Compare the processed event rate in BD FACSDiva software with the threshold counter on the cytometer. If the software event rate is much lower, quit and then restart the application.
Page 235 Window extension set Adjust the window extension, if expected in gated incorrectly needed. Refer to the population BD FACSDiva Software Reference Manual for information. Laser delay set incorrectly Adjust the laser delay settings. See Cytometer Quality Control Using BD FACSDiva Software on page 118.
Page 236 If necessary, adjust area scaling to make the Area measurement match the Height measurement. Cannot delete from Row not selected Select the row using the selection Parameters, Threshold, button. Compensation, or Ratio Data already recorded Create a new tube. tab views BD FACSAria User’s Guide...
Page 237: Fluidics Troubleshooting
Push firmly on each line to ensure it is connected. Fluidics cart air flow Air leak Contact your BD Biosciences <70 psi service engineer. Fluidics cart air flow Regulator not adjusted Contact your BD Biosciences >100 psi...
Page 238: Electronics Troubleshooting
Decrease the event rate or verify Overflow” in the threshold. Cytometer window Dirty flow cell Clean the flow cell. See Clean Flow Cell on page 184. “Cytometer not Unknown Perform the suggestions for a responding” in Status communication failure, above. BD FACSAria User’s Guide...
Page 239: Appendix A: Supplies And Options
This appendix provides a list of supplies and options that are available for the BD FACSAria cytometer. • To order spare parts and consumables from BD Biosciences from within the US, call (877) 232-8995 or go to bdbiosciences.com. Outside the US, contact your local BD Biosciences representative.
Page 240: Cytometer Supplies
Cytometer Supplies Optical Components The following filters and mirrors are mounted on the BD FACSAria cytometer. Use these part numbers if you need to order any replacement components. See Changing Filters on page 253 for instructions. Detector Array LP Mirror...
Page 241 The following filters and mirrors are provided with the violet-laser option. Detector Array LP Mirror Replacement Intended Dye (Laser) BP Filter Part No. Trigon 343797 (405-nm violet laser) ® 530/30 343798 Alexa Fluor ® 450/40 343801 Cascade Blue Pacific Blue™, Hoechst, DAPI, ®...
Page 242: Accessory Kit
Four-way 12 x 75-mm collection tube holder 333532 Two-way 12 x 75-mm collection tube holder 334904 Two-way 15-mL collection tube holder 345896 O-ring for collection-tube holder 337897 Adapter tray for microscope slides 335630 ACDU splash shield 334909 BD FACSAria User’s Guide...
Page 243 Item Part No. Spare nozzles: • 70 micron 342908 • 100 micron 342909 Nozzle locking lever (spring and plunger included) 337579 Nozzle O-rings (bag of 30; replacements sold individually) 333084 (each) Magnifying glass 33759907 Sample injection tubing (three 12-inch lengths) 335598 HPLC valve tubing (three 7-inch lengths) 335599...
Page 244: Other Replacement Parts
Auxiliary sensor probe (1-level) 334911 Air filter for plenum cabinet (side door) 334351 Air filter for ACDU cabinet (set of 3) 334821 ULPA filter and tubing replacement kit 334822 (set of 3, for use with the AMO) BD FACSAria User’s Guide...
Page 245: Laser Specifications
Laser Specifications The following class 3B lasers are mounted on the BD FACSAria cytometer. Wavelength Power Manufacturer Model (nm) (mW) Coherent Sapphire 488-20 JDS Uniphase 1144-P Point Source IFlex 2000-P-1-405- 0.65-10 StokerYale/Lasiris SNF-701L-660-35-1 deg single-line laser Appendix A: Supplies and Options...
Page 246: Consumables
• 559123 Particles, 3.0–3.4 μm (8 peaks) • 556286 • 556291 (brightest peak in 556286) BD FACS Accudrop beads BD Biosciences 345249 BD Cytometer Setup and Tracking BD Biosciences 641319 (CS&T) beads (1 vial) 642412 (3 vials) BD FACSAria User’s Guide...
Page 247: Reagents
(800) 438-2209 Sigma (800) 325-3010 BD FACS™ lysing solution BD Biosciences 349202 a. Refer to the BD Biosciences Immunocytometry Products Catalog or the BD Biosciences website (bdbio- sciences.com). b. US Patent Nos. 4,654,312; 4,902,613; 5,098,849 Appendix A: Supplies and Options...
Page 248: Labware
Bio-Rad Laboratories 223-9391 (800) 424-6723 (1,000 per box) 5-mL polystyrene test tubes, BD Biosciences 12 x 75-mm (BD Falcon™) • uncapped, 125 per bag • 352052 • capped, 125 per bag • 352054 • capped, 25 per bag • 352058 •...
Page 249: Cytometer Options
Cytometer Options Your BD FACSAria cytometer can be upgraded with the following options. Contact your sales representative for more information. Third-laser option The third-laser option upgrades your cytometer with a violet laser, a trigon, and two PMTs for greater flexibility in analysis and sorting.
Page 250: Application Options
Application Options The octagon and trigon detector arrays in the BD FACSAria cytometer allow many different combinations of mirrors and filters. The following screens show examples of how the collection optics are configured for a two- and a three-laser system.
Page 251: Three-Laser System
Three-Laser System This is a typical configuration for a three-laser system. Appendix A: Supplies and Options...
Page 252 Defining a Custom Configuration BD FACSDiva software is provided with initial configurations specific to your cytometer. Additional custom configurations can be defined. Refer to the BD FACSDiva Software Reference Manual and the Cytometer Setup and Tracking Application Guide for instructions. To switch between predefined configurations, select the appropriate configuration name and click Set Configuration.
Page 253 695/40 filter with the 675/20 filter to detect PerCP. If you want to install a custom filter or dichroic, the filter should comply with the following specifications. Table A-1 BD FACSAria filter specifications Filter Characteristic Dichroic LP Filters BP Filters Diameter 0.622 ±0.003 in.
Page 254 To remove the filter, pull it out of the slot. Figure A-2 Removing the FSC ND filter FSC ND filter To reinstall the filter, slide it into the slot with the filter side down. BD FACSAria User’s Guide...
Page 255 Appendix A: Supplies and Options...
Page 256 BD FACSAria User’s Guide...
Page 257: Appendix B: Application Settings
Appendix B Application Settings This appendix contains information on creating application settings. You can also refer to the Cytometer Setup and Tracking Application Guide for more information.
Page 258 Create a new experiment. Select the Cytometer Settings in the Browser. In the Inspector window, select the Parameters tab, and delete unwanted parameters. Click in the H checkbox to select Height for each parameter. See Figure B-1. BD FACSAria User’s Guide...
Page 259 Figure B-1 Inspector Window - Parameters tab Adjusting Area Scaling The required area scaling factor changes based on sheath pressure and particle size. The area scaling factors should be verified for each experiment performed on the instrument. In the Browser, right-click Global Sheet 1 and select Apply Analysis Template.
Page 260 Adjust the P1 gate around the population of interest. See Figure B-3. Figure B-3 FSC and SSC Plot Adjust the FSC area scaling. NOTE Click the Laser tab in the Cytometer window. See Figure B-4. Figure B-4 Adjusting FSC Area Scaling BD FACSAria User’s Guide...
Page 261 Adjust the FSC area scaling factor until the FSC-A signal matches the FSC-H signal. See Figure B-5. • Increase the area scaling factor if the APC-A signal is lower than APC-H. • Decrease the area scaling factor if the APC-A signal is higher than APC-H.
Page 262 A second global sheet is added with the plots created according to your selections in the parameters tab. See Figure B-7. Use the gray boxes and crosshairs to guide your optimization. Figure B-7 Optimizing PMT voltages BD FACSAria User’s Guide...
Page 263 Install the unstained control tube onto the cytometer. In the Cytometer window, optimize the settings for your application. • Optimize the FSC and SSC voltages to place the population of interest on scale. • Optimize the FSC threshold value to eliminate debris without interfering with the population of interest.
Page 264 Settings > Save, to save the values for reuse. See Figure B-8. Figure B-8 Saving Application Settings In the Applications Settings dialog, rename the application settings with a descriptive name. Click OK. The application settings are saved to the catalog. BD FACSAria User’s Guide...
Page 265 Index Numerics sort layouts 98, 165 sort populations 98, 166 4-Way Purity mode 75 adjusting amplitude 66, 110 area scaling 124, 259 Drop 1 66, 111 aborts drop delay 159 See also conflicts, sort. flow rate 48, 92 electronic 235 Home location 171 Access Stage button 101 laser delay 128...
Page 266 142 fluidics cart 22 printing 149 workstation 44 sorting 147, 155, 164 BD FACSDiva software See software. application settings beads about 258 Accudrop 159 adjusting area scaling 259 calibration 246 creating 258 beam splitters 54 optimizing PMT voltages 262...
Page 267 22 BD FACSAria system 21 chambers BD FACSDiva workspace 80, 92 ACDU 35 cytometer 26 cleaning bulk injection 191 electronics 42 sample injection 29 fluidics 27 sort block 32 optics 36 sort collection 35 workstation 44 changing computer...
Page 268 100 default configurations 250 sort precision modes 77 disconnect error 238 detectors 39, 52, 58 doors 26 devices, sorting 96 electronics 42 diode laser 41, 69 fluidics 27, 46 discriminating filters 55 not responding 238 BD FACSAria User’s Guide...
Page 269 105 satellites 111 exporting sort reports 104, 168 Drop 1 about 64, 66 adjusting 66, 111 FACSAria See BD FACSAria. drop delay, automatic method 162 FACSDiva software See software. ferrules, removing 201 fiber optics 37 editing filling containers 114...
Page 270 123, 136 nozzle 205 forward scatter (FSC) plates 170 detector 58 sample tubes 29 ND filter 241, 254 slides 170 removing 254 splash shield 169 frequency, drop drive 65 instrument FSC See forward scatter. See cytometer. BD FACSAria User’s Guide...
Page 271 interrogation point 30 unscheduled 205 managing aerosols 34 Masks about 71 labels default precision modes 75 parameter 143 Phase 74 precaution xviii Purity 73 label-specific tubes 138 Yield 71 laser delay, adjusting 122 Master DAQ overflow error 238 lasers micrometer dial 41, 160, 163 about 36 mirrors, dichroic 54 delay 61, 128...
Page 272 Yield Masks, using with 72, 75 primary laser, verifying area scaling 124 photodiodes 58 priming fluids 115, 185 photomultiplier tubes (PMTs) printing about 39, 58 sort reports 104, 168 applying voltages 60 worksheets 149 assigning 58, 87, 252 pulse, electronic 59 BD FACSAria User’s Guide...
Page 273 purging filters 198, 214 flow 48 Purity Masks 73 injection chamber 29 Purity mode 75, 165 interrogation 30 line backflush 183 changing 200 quality control (QC) pressure 48 about 119 temperature 83, 109 beads 122 samples, running 146 experiment 105 satellites, drop 111 log 151 Save Conflicts 99, 166...
Page 274 101 sort reports stream about 103 centering 68, 157 exporting 104, 168 control 65 printing 104, 168 deflecting 68 sort setup values 94, 95 flow rate 47 sorting starting 109 about 62, 153 troubleshooting 110, 220 BD FACSAria User’s Guide...
Page 275 viewing 41 sort strobe lens, cleaning 211 conflict rate 229 supplies, cytometer 240 counters 228 Sweet Spot results 229 about 63, 64 sorting 225 control 65 stream 110, 220 threshold 236 tubes adding sort layouts 98, 165 tanks See containers. agitating 83 target events 98, 166 compensation 138...
Page 276 Side Stream 67 worksheets printing 149 viewing 144 workspace components 80, 92 setting up 136 workstation about 44 shutting down 180 starting 109 Yield Masks about 71 Phase Masks, using with 72, 75 Yield mode 76 BD FACSAria User’s Guide...

BD FACSAria User Manual

About this Guide

Safety and Limitations

Chapter 1: BD Facsaria Cytometer Components

Chapter 2: Theory of Operation

Chapter 3: Using BD Facsdiva Software

Chapter 4: Running Samples

Chapter 5: Sorting

Chapter 6: Shutdown and Maintenance

Chapter 7: Troubleshooting

Appendix A: Supplies and Options

Appendix B: Application Settings

Quick Links

Troubleshooting

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