Aria 8.0 User's Guide

BD BiosciencesSan Jose, CA 95131-1807USATel (877) 232-8995Fax (800) 325-9637Brazil Tel ( 55) 11 -5 185 -9 99 5Fax (55) 11-5185-9895Europe Tel (32)

x BD FACSAria II User’s Guide Draft #5.1, October 2007Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

100 BD FACSAria II User’s Guide Sort MenuSelect commands in the Sort menu for the following:•Sort Setup—Downloads the most recently used settings for

Chapter 3: Using BD FACSDiva Software 101Sort SetupSort setup values for four different nozzle sizes can be downloaded using the Sort > Sort Setup

102 BD FACSAria II User’s Guide Table 3-2 Default Sort Setup valuesSetting 70 micron 85 micron 100 micron 130 micronSheath Pressure 70 45 20 10Ampli

Chapter 3: Using BD FACSDiva Software 103Sort LayoutThe Sort Layout window contains all sorting instructions and controls. The sort layout designates

104 BD FACSAria II User’s Guide Figure 3-9 Sort layout for collection tubes (top) and for a 48-well plate (bottom)Figure 3-10 Sort layout for a fro

Chapter 3: Using BD FACSDiva Software 105Setting Up a Sort Layout Sort layouts can be added to tubes or global worksheets. •Create tube-specific layou

106 BD FACSAria II User’s Guide •Ask User—Prompts the user each time the sort is stopped to select whether or not to save the sort report. This is th

Chapter 3: Using BD FACSDiva Software 107•For a four-tube layout, conflicts for the Far Left tube are sorted to the left, and conflicts for the Far Ri

108 BD FACSAria II User’s Guide Click the Sort button again to stop sorting before reaching the requested number of events. The counters stop at the

Chapter 3: Using BD FACSDiva Software 109• Sort Rate—number of events per second that met the sort criteria and were sorted• Conflict Count—number of

Draft #5.1, October 2007 Contents xiUsing the BD Temperature Control Option . . . . . . . . . . . . . . . . . . . . . . . . . 303Setting Up the Wate

110 BD FACSAria II User’s Guide Sort ReportSelect Sort > Sort Reports to view all of the saved reports for the current sort layout (see Figure 3-1

Chapter 3: Using BD FACSDiva Software 111Figure 3-14 Typical Sort ReportThe Sort Report window contains a File menu where you can select to print or

112 BD FACSAria II User’s Guide TemplatesWhen you install BD FACSDiva software for the BD FACSAria II cytometer, the following additional experiment

1134Running SamplesThe following topics are covered in this chapter:•Cytometer Startup on page114•Checking Cytometer Performance on page130•Applicatio

114 BD FACSAria II User’s Guide Cytometer StartupFollow these steps to start up your BD FACSAria II cytometer.1 Start up the workstation.2 Turn on th

Chapter 4: Running Samples 1155 Check the fluidics levels in the Cytometer window. Replenish fluids or empty the waste, if needed.To display the Cytom

116 BD FACSAria II User’s Guide 2 Verify that the air and fluid lines are disconnected from the ethanol tank and connected to the sheath tank, then c

Chapter 4: Running Samples 1174 Remove the closed-loop nozzle from the flow cell, then click Done.5 Insert the correct nozzle size in the flow cell.a

118 BD FACSAria II User’s Guide Starting the StreamThe system can take anywhere from 20 seconds to several minutes to reach the correct pressure and

Chapter 4: Running Samples 119Setting Up the BreakoffEstablishing a stable drop pattern in the breakoff window is an important step in getting optimal

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120 BD FACSAria II User’s Guide 2 Verify that the small satellite droplets are merging with the large droplets.Satellite-merging is largely dependent

Chapter 4: Running Samples 121NOTE The breakoff patterns in Table 4-1 are intended as examples. The patterns will not always look exactly like these.T

122 BD FACSAria II User’s Guide Setting Up the Fluidics CartCheck the fluid levels in the sheath tank and waste containers every time you use the cyt

Chapter 4: Running Samples 123NOTE Make sure the blue sheath fluid line between the sheath tank and the cytometer does not come into contact with anyt

124 BD FACSAria II User’s Guide 5 Fill the tank with sheath fluid up to the upper weld line on the inside of the tank. See Figure 4-5.NOTE Do not ove

Chapter 4: Running Samples 1258 Connect the air line. The system is ready to run again.Refilling the Ethanol Shutdown Tank The fluid level in the stai

126 BD FACSAria II User’s Guide 6 Replace the cover and tighten the knob. Make sure the large O-ring on the inside of the cover is seated correctly a

Chapter 4: Running Samples 1274 Replace the container cap and hand-tighten it until it is fully closed.5 If you disconnected the sensor and quick-rele

128 BD FACSAria II User’s Guide Figure 4-9 Waste container details1 Disconnect the waste container’s sensor and fluid line connectors from their res

Chapter 4: Running Samples 129Figure 4-10 Draining liquid from the trap3 Empty the waste container according to your standard laboratory procedures f

xiiiAbout This GuideThis user’s guide contains the instructions necessary to operate and maintain your BD FACSAria™ II flow cytometer. Because many in

130 BD FACSAria II User’s Guide Checking Cytometer Performance Before setting up an experiment, you should first run a performance check. A performan

Chapter 4: Running Samples 131Figure 4-11 Main CS&T window2 Verify that the bead lot information under Setup Beads matches the Cytometer Setup an

132 BD FACSAria II User’s Guide 3 Verify that the cytometer configuration is correct for your experiment.If the cytometer is not set to the correct c

Chapter 4: Running Samples 1334 Verify that the current configuration has a valid baseline defined.If not, refer to the Cytometer Setup and Tracking A

134 BD FACSAria II User’s Guide Running a Performance CheckThe performance check feature of Cytometer Setup and Tracking will check the cytometer’s d

Chapter 4: Running Samples 1354 Select File > Exit to close the Cytometer Setup and Tracking window and connect back to the BD FACSDiva interface.5

136 BD FACSAria II User’s Guide Application SettingsApplication settings are associated with a cytometer configuration and include the parameters nee

Chapter 4: Running Samples 1375 Select the H checkbox to select Height for each parameter. See Figure 4-12 on page 137.Figure 4-12 Inspector Window -

138 BD FACSAria II User’s Guide 6 Adjust the FSC and SSC voltages to place the particles on scale. See Figure 4-13 on page 138.Figure 4-13 Adjusting

Chapter 4: Running Samples 139Figure 4-15 Adjusting FSC Area Scalingb Adjust the FSC area scaling factor until the FSC-A signal matches the FSC-H sig

xiv BD FACSAria II User’s Guide ConventionsThe following tables list conventions used throughout this guide. Table 1 lists the symbols that are used

140 BD FACSAria II User’s Guide Figure 4-16 FSC area scaling comparison9 Adjust the blue laser area scaling until the FITC-A signal matches the FITC

Chapter 4: Running Samples 141Figure 4-17 Deselecting Height parameterOptimizing PMT Voltages 1 Right-click Cytometer Settings in the Browser, then s

142 BD FACSAria II User’s Guide b Optimize the FSC threshold value to eliminate debris without interfering with the population of interest.c If neede

Chapter 4: Running Samples 143Saving Application Settings1 Right-click Cytometer Settings in the Browser, then select Application Settings > Save,

144 BD FACSAria II User’s Guide Data CollectionBefore you record data for a sample, cytometer settings should be optimized to position the cells of i

Chapter 4: Running Samples 1453 Make sure the octagon and trigon(s) contain appropriate filters.For assistance, see Cytometer Configuration on page 84

146 BD FACSAria II User’s Guide 7 Rename the experiment appropriately (for example, 4-Color experiment).8 Right-click the experiment level Cytometer

Chapter 4: Running Samples 147Figure 4-21 Example mismatch error message10 Select Experiment > Compensation Setup > Create Compensation Control

148 BD FACSAria II User’s Guide conjugates due to lot-to-lot variation. Refer to the BD FACSDiva Software Reference Manual for more information about

Chapter 4: Running Samples 149Figure 4-22 Voltages adjusted5 Click the Threshold tab and adjust the FSC threshold, if needed.Set the threshold to rem

About This Guide xvTechnical AssistanceFor technical questions or assistance in solving a problem:•Read the section of the user’s guide specific to th

150 BD FACSAria II User’s Guide 12 Verify that the Snap-to Interval gate encompasses the positive population (Figure 4-23). Adjust the gate, if neede

Chapter 4: Running Samples 151Data Recording and AnalysisOnce you have optimized the cytometer electronics for your sample type, you are ready to reco

152 BD FACSAria II User’s Guide Setting Up the ExperimentBefore you record data, set up an experiment with appropriate tubes, plots, and labels for y

Chapter 4: Running Samples 153Setting Up the Global WorksheetA global worksheet is used to perform doublet discrimination and to set up plots to previ

154 BD FACSAria II User’s Guide 3 Turn on biexponential display.a Select the two plots.b In the Inspector, select the checkboxes for X Axis and Y Axi

Chapter 4: Running Samples 155Recording DataThis section describes how to adjust the gates to eliminate doublets and record singlet events.1 Move the

156 BD FACSAria II User’s Guide 10 Repeat steps 7 through 9 for the remaining tubes.Analyzing DataThis section describes how to set up plots, gates,

Chapter 4: Running Samples 1575 Right-click either fluorescence plot and select Create Statistics View.A statistics view is added to the worksheet.6 R

158 BD FACSAria II User’s Guide Figure 4-26 Sample analysis for mixed-bead tube

Chapter 4: Running Samples 159Performing a Batch AnalysisBatch analysis allows you to automatically advance through a selected set of tube data when u

xvi BD FACSAria II User’s Guide LimitationsThis instrument is for Research Use Only. Not for use in diagnostic or therapeutic procedures.BD Bioscienc

160 BD FACSAria II User’s Guide • Select the Statistics checkbox to export the statistics to a separate file, then enter a name for the statistics fi

1615SortingYou can program BD FACSDiva software to sort a specified number of particles from multiple populations into a variety of sorting devices in

162 BD FACSAria II User’s Guide Before you begin, you should be familiar with BD FACSAria II operation using BD FACSDiva software. Review chapters 2

Chapter 5: Sorting 1637 Install the required collection device and set up the side streams.See Setting Up for Bulk Sorting on page 164 or Setting Up f

164 BD FACSAria II User’s Guide Setting Up for Bulk SortingThis section describes how to set up the streams for two- or four-way sorting. For sorting

Chapter 5: Sorting 165Figure 5-1 Turning on the deflection platesMake sure the center stream image does not move after the plates are turned on. Majo

166 BD FACSAria II User’s Guide If you cannot see a stream image or the image is dim, adjust the micrometer dial on the diode laser to better view th

Chapter 5: Sorting 167NOTE Before beginning these procedures, make sure the stream is stable and the Sweet Spot is on. Setting Up the ExperimentThe st

168 BD FACSAria II User’s Guide Using Manual Drop DelayThis section describes the manual method of optimizing the drop delay. 1 Load a tube filled wi

Chapter 5: Sorting 169There is no need to collect the beads. When the drawer is closed, the beads are sorted to waste.6 Adjust the micrometer dial (se

171Cytometer ComponentsThe BD FACSAria II flow cytometer is a high-speed fixed-alignment benchtop cell sorter. The cytometer can be operated at varied

170 BD FACSAria II User’s Guide Figure 5-3 Sorting Accudrop beads in Initial mode10 In the Sort Layout window, change the precision mode to Fine Tun

Chapter 5: Sorting 171Determining the Drop Delay – Automatic MethodThe Auto Drop Delay feature automates setting the drop delay to get optimized resul

172 BD FACSAria II User’s Guide 5 Click Cancel at the Confirm dialog.There is no need to collect the beads. When the drawer is closed, the beads are

Chapter 5: Sorting 173Figure 5-5 Auto drop delay dialogSortingBefore beginning the sort, do the following:1 Perform the steps outlined in Setting Up

174 BD FACSAria II User’s Guide Setting Up the Experiment;Tip When more than one drop is deflected in the same direction, residual charge from th

Chapter 5: Sorting 175• Select the sort location field(s) to be sorted into. Select multiple fields by dragging the mouse. Select a row or column by c

176 BD FACSAria II User’s Guide Starting and Monitoring the Sort1 Open the sort collection chamber door and install the collection tubes, plate, or s

Chapter 5: Sorting 177; Tip Click Record Data to save data for the tube. Acquisition and sorting continue when the required number of events has b

178 BD FACSAria II User’s Guide Pausing and Resuming a SortThe Pause/Resume feature allows you to temporarily pause the sort, and still retain the co

Chapter 5: Sorting 179Setting Up for Sorting Into a Plate or SlideThe following sections describe how to set up for sorting into a plate or slide. For

18 BD FACSAria II User’s Guide Figure 1-1 BD FACSAria II cytometer components Fluidics CartA separate fluidics cart supplies sheath and cleaning flu

180 BD FACSAria II User’s Guide 2 Click the Access Stage button to bring the ACDU stage to the front.a Open an experiment, if one is not already open

Chapter 5: Sorting 181Figure 5-7 Sort order on a slideSetting Up the StreamThis section describes how to optimize side stream deflection and how to a

182 BD FACSAria II User’s Guide 2 Turn on the deflection plates.Click the Voltage button ( ) in the Side Stream window. The voltage warning light ill

Chapter 5: Sorting 183The stage moves to the pre-programmed Home position.6 Double-click the Test Sort button to deposit a drop at the Home location.7

184 BD FACSAria II User’s Guide Creating a Custom DeviceYou can program the ACDU stage to sort into any grid configuration. Create a custom device by

Chapter 5: Sorting 1853 Select the text in the Name field and enter a new name.4 Enter the number of sort location rows and columns.A device can have

186 BD FACSAria II User’s Guide Deleting a Custom Device1 Select Sort > Custom Devices.2 Select the name of the custom device to be deleted in the

1876Shutdown and MaintenanceThe BD FACSAria II cytometer is designed to require minimum maintenance. However, to preserve the reliability of the cytom

188 BD FACSAria II User’s Guide Daily Shutdown In the BD FACSAria II system, the recommended daily shutdown procedure is to run the Clean Flow Cell c

Chapter 6: Shutdown and Maintenance 1894 When prompted, install a tube containing approximately 3 mL of 15% Contrad® 70 cleaning solution, then click

Chapter 1: Cytometer Components 19Figure 1-2 Fluidics cart containersThe fluidics cart connects directly to the flow cytometer unit via a power cord,

190 BD FACSAria II User’s Guide Fluidics ShutdownThe Fluidics Shutdown command can be used to perform an extensive cleaning if the system is used to

Chapter 6: Shutdown and Maintenance 191Verify that there is an O-ring in the closed-loop nozzle before installing it.Figure 6-1 Closed-loop nozzle in

192 BD FACSAria II User’s Guide Figure 6-2 Connecting air and fluid lines for shutdown procedure5 When prompted, install a tube containing 3 mL of 1

Chapter 6: Shutdown and Maintenance 1936 Click OK when you see a message informing you the system can be turned off. 7 Turn off the cytometer main pow

194 BD FACSAria II User’s Guide Scheduled MaintenanceFor optimal cytometer functioning, perform the following procedures according to the recommended

Chapter 6: Shutdown and Maintenance 195Sample Line BackflushAfter a sample tube is unloaded, the sample line tubing within the sample injection chambe

196 BD FACSAria II User’s Guide 2 Click Start to start the backflush.3 Click Stop to stop the backflush, or click Cancel to stop the backflush and cl

Chapter 6: Shutdown and Maintenance 1974 Click OK when the tank prime is complete. Prepare for Aseptic SortUse the Prepare for Aseptic Sort command wh

198 BD FACSAria II User’s Guide To run t he Prepare fo r Aseptic Sort command:1 Select Cytometer > Cleaning Modes > Prepare for Aseptic Sort.Fo

Chapter 6: Shutdown and Maintenance 1994 Disconnect the bleach fluid line from the DI water port, and connect it back to the bleach tank port, then re

© 2007 Becton, Dickinson and Company. All rights reserved. No part of this publication may be reproduced, transmitted, transcribed, stored in retrieva

20 BD FACSAria II User’s Guide Figure 1-3 Fluidics cart power and fluid line connectors on cytometerConnecting to an External Air SupplyTo connect t

200 BD FACSAria II User’s Guide •To continue running samples, perform fluidics startup.•To turn off the system, perform the flow cell cleaning proced

Chapter 6: Shutdown and Maintenance 201Flow Cell Preventive Cleaning It is good practice to perform flow cell preventive cleaning once per month, or m

202 BD FACSAria II User’s Guide Changing Fluid FiltersWe recommend changing the fluid filters every 6 months. Spare filters are included with the acc

Chapter 6: Shutdown and Maintenance 203Changing the Sheath Filter We recommend changing the sheath filters every 3 months, or when increased debris in

204 BD FACSAria II User’s Guide Changing the Sample LinesThe primary sample line between the sample injection chamber and the pinch valve should be c

Chapter 6: Shutdown and Maintenance 205Changing the Primary Sample LineTo r epla ce the primary sample line, you will need a 12-inch length of replace

206 BD FACSAria II User’s Guide c Slide the pinch valve tubing on the inside of the teeth of the nut until it stops.d Couple both pieces of the fitti

Chapter 6: Shutdown and Maintenance 2074 Ensure that a cone-shaped ferrule is attached to the sample line.Figure 6-11 on page 206 shows an example of

208 BD FACSAria II User’s Guide Insert the sample line into the sample injection chamber fitting. Push the tubing from the top until it is slightly a

Chapter 6: Shutdown and Maintenance 209Flow Cell End1 Turn the stream off (if needed). 2 Unscrew the connecting nut at the top of the flow cell and sl

Chapter 1: Cytometer Components 21Power and OperationPower to the fluidics cart is supplied by the cytometer. The cart is activated when the cytometer

210 BD FACSAria II User’s Guide 6 Insert the pilot tubing into its fitting at the top of the flow cell, ensuring that the tubing reaches the intended

Chapter 6: Shutdown and Maintenance 211Changing the Air FiltersThe BD FACSAria II cytometer has two air filters: one in the sort collection chamber do

212 BD FACSAria II User’s Guide Changing the Sheath Tank Air Filter Check the inline air filter on the sheath tank air line periodically for any sign

Chapter 6: Shutdown and Maintenance 213Unscheduled MaintenanceThere are several cytometer components that should be cleaned periodically or checked fo

214 BD FACSAria II User’s Guide Changing NozzlesAs a general guideline, the nozzle size should be at least three times the diameter of the particle t

Chapter 6: Shutdown and Maintenance 215Use the magnifying glass in the accessory kit to make sure the O-ring is installed in the groove at the end of

216 BD FACSAria II User’s Guide Cleaning the NozzleUse the following procedure to clean the nozzle when the stream appears blocked or distorted. To v

Chapter 6: Shutdown and Maintenance 217Do not wipe the nozzle with anything, because it could leave fibers or other contamination, or dislodge the O-r

218 BD FACSAria II User’s Guide Closed-Loop Nozzle Maintenance The closed-loop nozzle and related tubing should be cleaned as needed if there are any

Chapter 6: Shutdown and Maintenance 219Sonicate the nozzle in a test tube containing DI water or a mild detergent. Repeat the sonication as needed unt

22 BD FACSAria II User’s Guide • Scheduled Maintenance on page 194•Fluidics Troubleshooting on page251Flow CytometerThe benchtop flow cytometer conta

220 BD FACSAria II User’s Guide 4 Insert the tubing into the union fitting and slowly tighten the nut until secure. Do not over-tighten. Pull gently

Chapter 6: Shutdown and Maintenance 2213 Loosen the sample line fitting nut at the top of the injection chamber to allow the sample line to slide free

222 BD FACSAria II User’s Guide 6 Pull the sample line up to operation height, slightly above the chamber viewing window.7 Place a tube onto the load

Chapter 6: Shutdown and Maintenance 223Changing the Pinch Valve Tubing The tubing that runs through the pinch valve should be changed as needed. The s

224 BD FACSAria II User’s Guide Figure 6-26 Tubing removed from pinch valve3 Unscrew the nut on the black collet fitting at each end of the tubing,

Chapter 6: Shutdown and Maintenance 225Figure 6-27 Collet nut fitting on pinch valve tubing 5 Install the new pinch valve tubing into the slot in the

226 BD FACSAria II User’s Guide Figure 6-28 Lower camera and diode laser windowsUpper Camera WindowClean the strobe lens and upper camera window whe

Chapter 6: Shutdown and Maintenance 2273 Open the sort block door. 4 Place 1–2 drops of DI water or ethanol on a cotton swab.5 Click the Breakoff wind

228 BD FACSAria II User’s Guide Removing the Deflection Plates You can remove the deflection plates for cleaning by pulling the plates out using the

Chapter 6: Shutdown and Maintenance 229Lubricating the Sample Injection Chamber O-Ring The O-ring at the bottom of the sample injection chamber should

Chapter 1: Cytometer Components 23See the following sections for more information about the flow cytometer:• Fluidics Components on this page• Optics

230 BD FACSAria II User’s Guide Using Custom Optical FiltersIf you want to install a custom filter or dichroic, the filter should comply with the fol

Chapter 6: Shutdown and Maintenance 231Cleaning the Optical FiltersOptical filters should be inspected occasionally and cleaned as necessary. The freq

232 BD FACSAria II User’s Guide Removing or Installing the FSC ND FilterFor applications involving large particles where events appear off scale on t

2337TroubleshootingThe tips in this section are designed to help you troubleshoot your experiments. Additional troubleshooting information can be foun

234 BD FACSAria II User’s Guide Troubleshooting the StreamObservation Possible Causes Recommended SolutionsStream not in center of aspiratorDifferenc

Chapter 7: Troubleshooting 235No stream or dripping streamNozzle inserted improperly Turn off the stream. Remove the nozzle. See Changing Nozzles on p

236 BD FACSAria II User’s Guide No stream when Stream control clickedSheath tank low or empty Refill the sheath tank. See Refilling the Sheath Tank o

Chapter 7: Troubleshooting 237Leaking or spraying around nozzleDefective or damaged nozzle O-ringReplace the O-ring. Nozzle inserted improperly Turn o

238 BD FACSAria II User’s Guide Troubleshooting the BreakoffUse the following examples to help troubleshoot problems with the breakoff image. Abnorma

Chapter 7: Troubleshooting 239 Sorting TroubleshootingObservation Possible Causes Recommended SolutionsUnstable breakoff while Sweet Spot is engagedRe

24 BD FACSAria II User’s Guide Figure 1-7 Main fluidics componentspinch valvesample injection chambercuvette flow cellsort block doorsort collection

240 BD FACSAria II User’s Guide Center stream is off center when the plate voltage is turned onVoltage center too low or too highAdjust the Voltage C

Chapter 7: Troubleshooting 241No deflection or insufficient deflectionInsufficient voltage • Increase the side-stream voltages using the slider contro

242 BD FACSAria II User’s Guide Fanning around center or side streamsNozzle inserted improperly Turn off the stream. Remove the nozzle and ensure tha

Chapter 7: Troubleshooting 243Unexpected sort results Incorrect drop delay Reset the drop delay. See Determining the Drop Delay – Manual Method on pag

244 BD FACSAria II User’s Guide Acquisition TroubleshootingObservation Possible Causes Recommended SolutionsNo events in plots after clicking Load

Chapter 7: Troubleshooting 245No events in plots after clicking Acquire Data (continued)Sample is not mixed properly Increase the Sample Agitation rat

246 BD FACSAria II User’s Guide Low area signal Area scaling is too low Adjust area scaling for the corresponding laser. See Cytometer Quality Contro

Chapter 7: Troubleshooting 247Erratic event rate Sample aggregates Filter the sample.Bulk injection O-ring is worn Contact your BD Biosciences service

248 BD FACSAria II User’s Guide Unexpectedly low event rate (continued)Sample line installed incorrectlyVerify the sample line installation. See Chan

Chapter 7: Troubleshooting 249Excessive amount of debris in plotsThreshold channel is too low Increase the threshold channel. See Calculating Compensa

Chapter 1: Cytometer Components 25Sample Injection ChamberThe sample injection chamber is where sample is introduced into the flow cytometer. During a

250 BD FACSAria II User’s Guide Increasing threshold results in decreased Area signalWindow extension is too low Slightly increase the window extensi

Chapter 7: Troubleshooting 251 Fluidics TroubleshootingObservation Possible Causes Recommended SolutionsNo fluid in line during system primeAir lock i

252 BD FACSAria II User’s Guide Electronics TroubleshootingObservation Possible Causes Recommended Solutions“Cytometer Disconnected” in Cytometer win

2538Technical Specifications•Cytometer Specifications on page254•Fluidics Cart Specifications on page259

254 BD FACSAria II User’s Guide Cytometer SpecificationsEnvironmentTable 8-1 Cytometer SpecificationsCytometer dimensions Height: 71 cm (28 in.)Widt

Chapter 8: Technical Specifications 255PerformanceTable 8-3 Performance SpecificationsFluorescence Resolution Coefficient of variation PI–Area of <

256 BD FACSAria II User’s Guide Sort PerformanceTable 8-4 Sort performance specificationsDrop Drive Frequency Range from 1 to 100,000 HzPurity and Y

Chapter 8: Technical Specifications 257Excitation OpticsLaser SpecificationsThe following class 3B lasers are mounted on the cytometer.NOTE For specif

258 BD FACSAria II User’s Guide Detector ArraysTable 8-7 Default setup for detector arraysDetector Array (Laser)PMT LP Mirror BP Filter Intended Flu

Chapter 8: Technical Specifications 259Fluidics Cart SpecificationsTable 8-8 Fluidics cart specificationsDimensions Height: 66 cm (26 in.)Width: 81cm

26 BD FACSAria II User’s Guide Tube HoldersA variety of tube holders are provided with the cytometer to accommodate tubes from 15-mL centrifuge tubes

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261Appendix ASupplies and ConsumablesThis appendix provides a list of supplies and options that are available for the BD FACSAria II cytometer. •To or

262 BD FACSAria II User’s Guide Cytometer SuppliesOptical ComponentsThe following filters and mirrors are mounted on the BD FACSAria II cytometer. Us

Appendix A: Supplies and Consumables 263The following filters and mirrors are provided with the violet-laser option.The FSC photodiode is provided wit

264 BD FACSAria II User’s Guide Accessory KitThe cytometer is shipped with an accessory kit containing the following items. Use these part numbers if

Appendix A: Supplies and Consumables 265Collet nut 19-66456-00Pinch valve tubing 641900Sample line filters (35 micron) 643152Sample line filters (50 m

266 BD FACSAria II User’s Guide Other Replacement PartsThe following items are not included in the accessory kit, but you can use the indicated part

Appendix A: Supplies and Consumables 267ConsumablesCytometer Setup ParticlesParticle Supplier Catalog No.BD Calibrite™ beads BD Biosciences•Two-color

268 BD FACSAria II User’s Guide ReagentsReagent Supplier Catalog No.BD FACSFlow sheath fluid BD Biosciences 340398(US and Latin America)342003(other

Appendix A: Supplies and Consumables 269LabwareItem Supplier Catalog No.1-mL microtubes Bio-Rad Laboratories(800) 424-6723223-9391 (1,000 per box)5-mL

Chapter 1: Cytometer Components 27Cuvette Flow CellThe cuvette flow cell is the heart of the BD FACSAria II cytometer (Figure 1-10). Within the flow c

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271Appendix BNear UV Laser OptionThe Near UV laser option for the BD FACSAria II system features a 375 nm, solid state laser that enables sorting and

272 BD FACSAria II User’s Guide System Laser ConfigurationsThe BD FACSAria II systems can be configured as two-laser, three-laser, or four-laser syst

Appendix B: Near UV Laser Option 273Specifications SafetyMake sure that everyone working with the system with the Near UV laser installed is familiar

274 BD FACSAria II User’s Guide OperationSelecting Optical FiltersWhen using the Near UV laser for side population studies, you can install a differe

Appendix B: Near UV Laser Option 275Creating a Custom ConfigurationThe Near UV laser uses the same PMT detectors as the violet laser, but typically wi

276 BD FACSAria II User’s Guide Switching From the Violet to the Near UV LaserThe system should be turned on and all cytometer startup procedures per

Appendix B: Near UV Laser Option 277Switching Back to the Violet LaserNOTE Depending on the application, the filters for the standard violet laser can

278 BD FACSAria II User’s Guide Side Population Application Guidelines The Near UV laser on the BD FACSAria II cytometer is very effective for use in

Appendix B: Near UV Laser Option 2796 Adjust the voltages so that the red blood cells are seen in the lower left corner and the dead cells line up on

28 BD FACSAria II User’s Guide NozzleThe BD FACSAria II cytometer is provided with four nozzle sizes (70, 85, 100, and 130 Pm) to accommodate a varie

280 BD FACSAria II User’s Guide TroubleshootingObservation Possible Causes Recommended SolutionsSignals dim or noisy Wrong laser turned on (Near UV i

281Appendix CBD Aerosol Management OptionThe BD™ aerosol management option (AMO) is a device that promotes the containment of aerosols by evacuating t

282 BD FACSAria II User’s Guide Option ComponentsThe BD Aerosol Management Option (AMO) includes the following:•An evacuator to generate negative pre

Appendix C: BD Aerosol Management Option 283Figure C-1 AMO evacuatorULPA FilterThe ULPA filter used in the BD AMO captures and retains 99.9999% of al

284 BD FACSAria II User’s Guide Operating the BD Aerosol Management OptionStarting Up the EvacuatorNOTE Before starting up the evacuator, make sure t

Appendix C: BD Aerosol Management Option 285Figure C-3 Installing the splash shield2 Ensure that an air filter is installed in the sort collection ch

286 BD FACSAria II User’s Guide Figure C-4 Turning on main power5 Press the POWER button on the membrane panel of the evacuator. 6 Press the up or d

Appendix C: BD Aerosol Management Option 287Figure C-5 Reading the filter flow gaugeSetting Up for SortingRefer to your instrument manual for instruc

288 BD FACSAria II User’s Guide Opening the Sort Collection Chamber Door 1 Use one of the following options to stop sample flow into the sort collect

Appendix C: BD Aerosol Management Option 289Turning Off the EvacuatorTurn off the evacuator after you have finished running biohazardous samples.1 Pla

Chapter 1: Cytometer Components 29Sort BlockAfter leaving the nozzle, particles pass through the sort block where they are either transported to waste

290 BD FACSAria II User’s Guide •Do not disconnect the tubing from the instrument manifold outlet or the ULPA filter unless you are changing the filt

Appendix C: BD Aerosol Management Option 291Figure C-6 Disconnecting tubing (side of instrument)3 Remove the spring-loaded filter hold-down.While pus

292 BD FACSAria II User’s Guide 4 Lift off the ULPA filter and attached tubing from the evacuator and dispose of both the filter and the tubing. 5 In

Appendix C: BD Aerosol Management Option 293Figure C-9 Seating the new filterNOTE For optimal evacuation of aerosols, the filter must be completely s

294 BD FACSAria II User’s Guide Figure C-10 Connecting new tubingNOTE For optimal evacuation of aerosols, ensure that the tubing is securely connect

Appendix C: BD Aerosol Management Option 295Figure C-11 Removing the air filter2 Install a new air filter in the door.Slide the new filter in from th

296 BD FACSAria II User’s Guide TroubleshootingThe tips in this section are provided to help you troubleshoot issues that arise when using the BD Aer

Appendix C: BD Aerosol Management Option 297Arrow keys not respondingImproper operation Push each button firmly before removing your finger from the c

298 BD FACSAria II User’s Guide Filter Flow Gauge TroubleshootingObservation Possible Cause Recommended SolutionZero reading on filter flow gaugePowe

Appendix C: BD Aerosol Management Option 299Specifications Specifications for the BD Aerosol Management Option are as follows.Evacuator• t7 CFM (ft3/m

FCC InformationWARNING: Changes or modifications to this unit not expressly approved by the party responsible for compliance could void the user’s aut

30 BD FACSAria II User’s Guide Deflection PlatesThe high-voltage deflection plates are used to deflect side streams during sorting. The plates are tu

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301Appendix DTemperature Control OptionThe BD™ temperature control option can be used to control the temperature of sorted samples in the BD FACSAria

302 BD FACSAria II User’s Guide Option Components The BD temperature control option includes the following:• A recirculating water bath• Specially de

Appendix D: Temperature Control Option 303Using the BD Temperature Control OptionNote that before you start the recirculating water bath, you must att

304 BD FACSAria II User’s Guide Figure D-2 Input and output ports on water bath5 Connect the insulated hoses from the recirculating water bath to th

Appendix D: Temperature Control Option 305NOTE Do not start up the water bath until after you have connected the recirculating water tubing, as descri

306 BD FACSAria II User’s Guide Figure D-4 Setting up the temperature control tube holderIf you need to remove the tubing, push in the orange collar

Appendix D: Temperature Control Option 307Setting Up the ACDU StageThis section describes how to attach the recirculating water tubing to the stage us

308 BD FACSAria II User’s Guide Figure D-5 Setting up the temperature control on ACDU stageIf you need to remove the tubing, push in the orange coll

Appendix D: Temperature Control Option 309Starting Up the Water BathNOTE To ensure that the sample collection device is at the correct temperature, st

Chapter 1: Cytometer Components 31Aerosol Management During sample acquisition and sorting, the sort block door and flow cell access door should be ke

310 BD FACSAria II User’s Guide 3 Wait at least 30 minutes (115 V model) or 60 minutes (110 V model) to allow the recirculating water reach the requi

Appendix D: Temperature Control Option 311Specifications Specifications for the recirculating water bath are as follows.NOTE The following specificati

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313Appendix EQC Using BD FACSDiva SoftwareThis appendix contains information on performing the quality control procedure using BD FACSDiva software.

314 BD FACSAria II User’s Guide Cytometer Quality Control Using BD FACSDiva SoftwarePerform cytometer quality control (QC) to ensure consistent perfo

Appendix E: QC Using BD FACSDiva Software 315The steps in this section show you how to set up the cytometer configuration in preparation for performin

316 BD FACSAria II User’s Guide Figure E-2 Error message showing detailsPreparing QC ParticlesPrepare QC particles, also known as beads, in a 12 x 7

Appendix E: QC Using BD FACSDiva Software 317This section describes how to optimize area scaling and laser delay using the QC template that is include

318 BD FACSAria II User’s Guide Figure E-3 Creating an experiment from the QC template4 Add the current month and year to the experiment name, renam

Appendix E: QC Using BD FACSDiva Software 319The loading port rises to enclose the tube within the chamber. Once the tube is loaded, acquisition start

32 BD FACSAria II User’s Guide Sort Collection ChamberCollection devices are installed in the sort collection chamber to collect sorted samples. The

320 BD FACSAria II User’s Guide 7 Adjust the FSC area scaling until the FSC-A signal matches the FSC-H signal, if needed.a Click the Laser tab in the

Appendix E: QC Using BD FACSDiva Software 321Figure E-4 FSC area scaling factor set incorrectly (left) and correctly (right)

322 BD FACSAria II User’s Guide 8 Adjust the FITC voltage to place the FITC-H signal at approximately 100 x 103.a Click the Parameters tab in the Cyt

Appendix E: QC Using BD FACSDiva Software 323Figure E-5 Blue laser area scaling before adjustment (left) and after (right) Adjusting Area Scaling and

324 BD FACSAria II User’s Guide The Sort Setup enters area scaling and laser delay values from the last time the Sort Setup was used. For this reason

Appendix E: QC Using BD FACSDiva Software 325e Reset the window extension to the appropriate setting (typically 2). 3 Adjust the red laser area scalin

326 BD FACSAria II User’s Guide 2 Adjust the PMT voltage for each parameter to set the signal to approximately 100 x 103. 3 Set the Flow Rate to 1.0

Appendix E: QC Using BD FACSDiva Software 327Figure E-6 Recorded QC data

328 BD FACSAria II User’s Guide Reusing the QC ExperimentThe QC experiment was designed so you can reuse it for subsequent QC runs by duplicating the

Appendix E: QC Using BD FACSDiva Software 329d Ensure that the red and violet (or Near UV) laser delays are set appropriately by setting the window ex

Chapter 1: Cytometer Components 33The sort collection chamber door should be kept closed when sorting into a plate. The door keeps the chamber free of

330 BD FACSAria II User’s Guide be addressed. To track cytometer performance over time, copy means and CVs for each parameter onto a QC log.You can a

331Numerics4-Way Purity mode 73AabortsSee also conflicts, sort.electronic 249Access Stage button 108accessory kit, contents 264Accudropabout 67experim

332 BD FACSAria II User’s Guideamplitudeabout 63adjusting 64, 119analysisdata 151printing 157sorting 156, 163, 173application settingsabout 136adjusti

Index 333changingair filters 211fluid filters 202nozzles 214optical filters 230sample lines 204waste cap 128charging drops 68circuit breaker 39cleanin

334 BD FACSAria II User’s GuidecontrolsSee also buttons.ACDU stage 108acquisition 97aspirator drawer 66, 108attenuation 66compensation 147cytometer (s

Index 335delaydrop 66, 67, 166See also Accudrop.laser 59, 323deletingcustom devices 186sort populations 107sort precision modes 75detectors 36, 50, 56

336 BD FACSAria II User’s Guidefiltersabout 51air, changing 211bandpass 53changing 202, 230default setup 258discriminating 53fluid, changing 202holder

Index 337hazardsmechanical 40heating samples 82, 114height parameters 58holderscollection tube 32, 164, 306optical filter 263sample tube 26Home Device

338 BD FACSAria II User’s GuideNNear UV laserabout 271configurations 272optical filters 274side population guidelines 278specifications 273neutral den

Index 339pinch valvechanging tubing 223collet fitting 225plate voltage 67platesdeflection 30, 164installing 180, 308sorting into 179plotsexcessive deb

34 BD FACSAria II User’s Guide Fiber optics direct the laser light in a precise and constant manner onto beam-shaping prisms, which in turn transmit

340 BD FACSAria II User’s Guidesampleagitation 81core diameter 47flow 46injection chamberabout 25lubricating O-ring 229interrogation 27linebackflush 1

Index 341Sort button 107sort layoutsabout 68, 103creating 105, 174custom 100, 184editing 107entering populations 105, 174sort precision modes4-Way Pur

342 BD FACSAria II User’s Guidestreamcentering 66, 166control 63deflecting 67flow rate 45starting 117troubleshooting 118, 234viewing 38strobe lens, cl

Index 343VView Configurations selection 84viewing global worksheets 153viewsSee also windows.sort layout 103voltageadjusting PMT 58controls 66warning

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Chapter 1: Cytometer Components 35Collection OpticsFrom the cuvette flow cell, laser light is collected by a fluorescence objective lens that is gel-c

36 BD FACSAria II User’s Guide Figure 1-18 Transmission pathways in an octagonDetectorsA standard system is equipped with an octagon containing six

Chapter 1: Cytometer Components 37Figure 1-19 Fully loaded detector arraysAt installation, the octagon and trigon arrays are set up with the filter a

38 BD FACSAria II User’s Guide Stream-Viewing OpticsThe BD FACSAria II cytometer is equipped with optical components that are used to view the stream

Chapter 1: Cytometer Components 39Cytometer ElectronicsThe electronic components consist of power controls and connectors along with processing boards

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40 BD FACSAria II User’s Guide Emergency Stop ButtonThe emergency stop button to the right of the loading port (Figure 1-22) is a safety feature that

Chapter 1: Cytometer Components 41Do not reset the button until the message appears. To do so, turn the button clockwise until the light turns off and

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432Theory of OperationThis chapter describes how the BD FACSAria II cytometer works and how BD FACSDiva software components are used to operate differ

44 BD FACSAria II User’s Guide Fluid MovementThe fluidics system is responsible for moving particles from the sample injection chamber through the cu

Chapter 2: Theory of Operation 45a constant pressure. You can view the current sheath pressure setting using the Cytometer > Sheath Pressure comman

46 BD FACSAria II User’s Guide Sample FlowSample is introduced into the cuvette when the Load button is clicked in the Acquisition Dashboard (Figure

Chapter 2: Theory of Operation 47Hydrodynamic FocusingIn the flow cell, pressurized sheath fluid surrounds the sample fluid to hydrodynamically focus

48 BD FACSAria II User’s Guide Signal GenerationThe following sections describe how signals are generated when cells or particles intercept the laser

Chapter 2: Theory of Operation 49Fluorescent SignalsWhen cells or particles stained with fluorochrome-conjugated antibodies or other dyes pass through

vContentsConventions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xivTechnical Assistance . . .

50 BD FACSAria II User’s Guide Signal DetectionFrom the cuvette flow cell, scattered and fluorescent light is collected by the fluorescence objective

Chapter 2: Theory of Operation 51FiltersOptical filters modify the spectral distribution of light scatter and fluorescence directed to the detectors

52 BD FACSAria II User’s Guide Longpass FiltersLongpass (LP) filters pass wavelengths longer than the filter rating and reflect shorter wavelengths.

Chapter 2: Theory of Operation 53Bandpass FiltersBandpass (BP) filters transmit a relatively narrow range or band of light. Bandpass filters are typic

54 BD FACSAria II User’s Guide Figure 2-10 Bandpass (BP) vs discriminating (DF) filtersIn the detector arrays, DF filters block high-intensity laser

Chapter 2: Theory of Operation 55Neutral Density FiltersNeutral density (ND) filters transmit a fixed percentage of light, reducing the transmitted in

56 BD FACSAria II User’s Guide DetectorsDetectors within each detector array convert light signals into electrical signals that can be processed by t

Chapter 2: Theory of Operation 57Electronic ProcessingAs cells or other particles pass through the focused laser beams, they scatter the laser light a

58 BD FACSAria II User’s Guide Pulse ParametersA parameter is a pulse property that is generated by a single photomultiplier tube or photodiode, meas

Chapter 2: Theory of Operation 59Laser DelaySample interrogation takes place within the cuvette flow cell. Fiber optic cables are used to direct laser

vi BD FACSAria II User’s Guide Draft #5.1, October 2007Detector Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

60 BD FACSAria II User’s Guide SortingDuring sorting, drop drive energy is applied to the stream to break it into highly uniform droplets. Droplets d

Chapter 2: Theory of Operation 61Figure 2-14 SortingDrop FormationThe BD FACSAria II cytometer constantly applies drop drive energy to the stream. Dr

62 BD FACSAria II User’s Guide Breakoff Window The upper camera transmits an image of the drop breakoff to the Breakoff window, where video image pro

Chapter 2: Theory of Operation 63Note that changes to values in the Sort Setup windows (Breakoff and Side Stream) are automatically saved. At startup,

64 BD FACSAria II User’s Guide Typically, when setting up for sorting, you use the Amplitude to set the required drop breakoff, and copy the generate

Chapter 2: Theory of Operation 65Side Stream FormationSide streams are formed when the voltage is on and you are sorting, or when you click Voltage, t

66 BD FACSAria II User’s Guide Table 2-2 Side Stream window controlsControl DescriptionVoltage button Turns the plate voltage on and off.Test Sort b

Chapter 2: Theory of Operation 67Drop Delay OverviewThe BD FACSAria II cytometer includes integrated Accudrop technology to assist in setting an accur

68 BD FACSAria II User’s Guide See Determining the Drop Delay – Manual Method on page 166 for more information.Auto Drop DelayThe Auto Drop Delay fea

Chapter 2: Theory of Operation 69Figure 2-17 Flow cell with stream-charging wireThe amount and type of charge determines where the drop will be sorte

Draft #5.1, October 2007 Contents viiStarting the Stream . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118Setting

70 BD FACSAria II User’s Guide Yield MaskThe Yield Mask setting defines how close to the edge of the drop, in 1/32-drop increments, a particle of int

Chapter 2: Theory of Operation 71Purity MaskThe Purity Mask setting defines how close, in 1/32-drop increments, a contaminating drop can be located be

72 BD FACSAria II User’s Guide Phase MaskParticles near the drop edge can affect the breakoff and alter the trajectory of the deflected drop. The Pha

Chapter 2: Theory of Operation 73Phase Masks cannot be used in conjunction with Yield Masks. Therefore, when the Phase Mask is greater than zero, the

74 BD FACSAria II User’s Guide to zero to ensure that residual charges from adjoining drops do not degrade the quality of side streams. The 4-Way Pur

Chapter 2: Theory of Operation 75Defining New Precision ModesDefault Precision modes cannot be edited or deleted. However, you can create new modes an

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773Using BD FACSDiva SoftwareMany BD FACSAria II cytometer functions are controlled using BD FACSDiva software. This chapter provides a general overvi

78 BD FACSAria II User’s Guide Workspace ComponentsWhen you start BD FACSDiva software, the workspace appears (Figure 3-1). For a general overview of

Chapter 3: Using BD FACSDiva Software 79Cytometer ControlsMost BD FACSAria II-specific cytometer controls are accessed through the Cytometer menu. Con

viii BD FACSAria II User’s Guide Draft #5.1, October 2007Starting and Monitoring the Sort . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

80 BD FACSAria II User’s Guide Fluidics ShutdownFluidics shutdown removes sheath from the lines and replaces it with ethanol, and cleans the cuvette

Chapter 3: Using BD FACSDiva Software 81Sample AgitationSelect Cytometer > Sample Agitation to specify the speed at which samples are agitated. You

82 BD FACSAria II User’s Guide Sample TemperatureUse the Sample Temperature command to set the temperature inside the sample injection chamber. You c

Chapter 3: Using BD FACSDiva Software 83Fluidics Level IndicatorsBD FACSDiva software provides fluidics level indicators in the Cytometer window (Figu

84 BD FACSAria II User’s Guide Cytometer ConfigurationThe menu selections shown in Figure 3-4 open the Cytometer Setup and Tracking (CS&T) module

Chapter 3: Using BD FACSDiva Software 85Cytometer Configuration WindowThe BD FACSAria II cytometer is equipped with a specific set of lasers, filters,

86 BD FACSAria II User’s Guide Figure 3-5 Example configuration for a three-laser systemBefore you start any experiment, verify that the cytometer c

Chapter 3: Using BD FACSDiva Software 87Selections in the Cytometer Configuration window determine which parameters are available for your experiment.

88 BD FACSAria II User’s Guide Cytometer Status ReportThe Cytometer Status Report provides a list of all cytometer settings at the time the report wa

Chapter 3: Using BD FACSDiva Software 89Figure 3-6 Cytometer Status Report

Draft #5.1, October 2007 Contents ixLubricating the Sample Injection Chamber O-Ring . . . . . . . . . . . . . . . . 229Using Custom Optical Filters

90 BD FACSAria II User’s Guide Custom ConfigurationsBefore you can record data, you must first ensure that the cytometer configuration is appropriate

Chapter 3: Using BD FACSDiva Software 91Adding Parameters, Filters, and MirrorsBefore creating a custom configuration, verify that the necessary param

92 BD FACSAria II User’s Guide Copying a Base ConfigurationYou cannot edit or delete your Base Configuration. However, you can use it as a starting p

Chapter 3: Using BD FACSDiva Software 936 Enter a descriptive name and press Enter.For example, use the name 70-70, meaning 70 micron nozzle at 70 psi

94 BD FACSAria II User’s Guide 2 To edit the nozzle size an d sheath pressure for this configuration: a Enter the appropriate sheath pressure value

Chapter 3: Using BD FACSDiva Software 95Verifying that the Configuration Matches the Sort SetupTo verify that the sheath pre ssure in the Sort Setup

96 BD FACSAria II User’s Guide The new sheath pressure is automatically saved with the current Sort Setup mode. ; Tip The name of the configurati

Chapter 3: Using BD FACSDiva Software 97Figure 3-7 CS&T Mismatch MessageAcquisition ControlsAlong with the controls described in the BD FACSDiva

98 BD FACSAria II User’s Guide •Load—Lifts a tube into the sample injection chamber, starts sample agitation (if agitation is turned on), and starts

Chapter 3: Using BD FACSDiva Software 99Figure 3-8 BD FACSDiva sorting controls Table 3-1 Description of sorting controlsThe Sort menu provides acce