Bioanalyzer CBA Flex Sets: Instrument Setup, Data ... · BD FACSArray™ Bioanalyzer BD™ CBA Flex...

BD FACSArray™ Bioanalyzer

BD™ CBA Flex Sets: Instrument Setup, Data Acquisition, and AnalysisInstruction Manual

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FCAP Array™ is a registered trademark of Soft Flow, Inc.

Unless otherwise specified, all products are for Research Use Only. Not for use in diagnostic or therapeutic procedures. Not for resale.

Table of Contents (continued)

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Table of Contents

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

. Monthly.Instrument.Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

. . Performing.Instrument.Setup. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

. . Transferring.the.Experiment.Template. . . . . . . . . . . . . . . . . . . . . . . . . . 4

. Routine.BD™.CBA.Flex.Set.Data.Acquisition.and.Analysis . . . . . . . . . . . . . 4

. . Creating.an.Experiment.in.FCAP.Array.Software. . . . . . . . . . . . . . . . . 4

. . Exporting.the.Experiment.File. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

. . Acquiring.Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

. . Analyzing.Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Monthly.BD.FACSArray™.Bioanalyzer.Setup. . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

. Creating.an.Instrument.Setup.Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . 5

. Preparation.of.Instrument.Setup.Beads. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

. . Adjusting.Loader.Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

. . Adjusting.Scatter.and.Far.Red.Voltages. . . . . . . . . . . . . . . . . . . . . . . . . 9

. . Adjusting.Voltages.with.Well.A4. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

. . Adjusting.Voltages.with.Well.A5. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

. . Correcting.for.Optical.Spillover . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

. Saving.the.Instrument.Setup.Experiment.as.a.Template. . . . . . . . . . . . . . . 15

. Transferring.the.Experiment.Template. . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Routine.BD™.CBA.Flex.Set.Data.Acquisition.and.Analysis. . . . . . . . . . . . . . . . 18

. Creating.an.experiment.using.the.FCAP.Array™.Software . . . . . . . . . . . . . 18

. Using.the.Experiment.Wizard. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

. Entering.Test.Samples.and.Dilutions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

. Creating.a.New.Plex. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

. Choosing.Plex.Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

. Specifying.Standards. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

. Creating.a.Plate.Layout . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

. Exporting.the.Experiment.File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

. Importing.Experiment.File.into.BD.FACSArray™.System.Software. . . . . . . 22

. Acquiring.Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

. . Sample.Preparation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

. Analysis.of.Sample.Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Troubleshooting.Tips. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

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IntroductionThe.BD.FACSArray™.bioanalyzer.has.been.designed.for.simple.setup.and.acquisition.of.the.BD™.Cytometric.Bead.Array.(CBA).Flex.Sets ..A.monthly.setup.must.be.performed.and.then.the.instrument.can.be.used.daily.for.the.acquisition.of.the.Flex.Set.assays ..The.BD.FACSArray.bioanalyzer.and.software.can.be.integrated.with.FCAP.Array™.software.to.encompass.the.entire.bead.assay.procedure,.from.instrument.setup.to.analysis.report ..

This.manual.describes.the.recommended.setup,.acquisition,.and.analysis.workflow.for.performing.BD™.CBA.Flex.Set.bead.assays.with.the.BD.FACSArray.bioanalyzer ..It.consists.of.the.main.steps.shown.in.the.following.figure.(steps.that.are.performed.using.BD.FACSArray.system.software.are.displayed.with.a.thicker.border) .

Set Up Instrument

TransferTemplate

ExportExperiment

ImportExperiment

AquireData

AnalyzeData

Create FCAPExperiment

These.main.steps.are.covered.in.the.following.sections:

Monthly Instrument SetupPerforming Instrument Setup

. •. Perform.instrument.setup.on.the.BD.FACSArray.bioanalyzer ..Save..the.instrument.setup.Experiment.as.an.Experiment.Template.in.the..BD.FACSArray.system.software ..

Transferring the Experiment Template

. •. Transfer.the.template.file.from.the.BD.FACSArray.templates.folder.to.the.FCAP Array Templates folder .

Routine BD™ CBA Flex Set Data Acquisition and AnalysisCreating an Experiment in FCAP Array™ Software

. •. Create.an.FCAP.Array™.software.Experiment.to.use.with.the..BD.FACSArray.bioanalyzer.to.acquire.your.BD.CBA.Flex.Set.assay ...Skip.the.Plex.cluster.assignment .

Exporting the Experiment File

. •. Export.the.Experiment.file.from.FCAP.Array.Software.into.the..BD.FACSArray.Software .

. •. Import.the.Experiment.file.to.the.BD.FACSArray.Software .

Acquiring Data

. •. Using.the.BD.FACSArray.bioanalyzer.and.software,.acquire.BD.CBA.Flex.Set.sample.data,.and.export.the.FCS.2 .0.list-mode.data.files .

Analyzing Data

. •. Using.FCAP.Array.software,.perform.plex.cluster.assignment,.and.assign.data.files.to.Experiment.samples ..Analyze.the.data,.review.the.results,.and.finalize.the.Experiment ..

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Monthly BD FACSArray™ Bioanalyzer SetupThe.monthly.15.minute.instrument.setup.procedure.described.in.this.section.creates.an.experiment.template.using.BD.FACSArray.system.software ..This.experiment.template.can.be.exported.to.FCAP.Array.software.to.prepare.a.BD.CBA.Flex.Set.experiment ..The.files.created.in.this.instrument.setup.procedure.are.used.to.ensure.the.proper.instrument.setup.and.compensation.is.applied.to.all.sample.files.collected.in.a.given.experiment ..

Note:. It.is.recommended.that.you.become.familiar.with.the..instrument.setup.procedure.prior.to.starting.a.BD.CBA.Flex.Set.experiment ..It.is.also.recommended.that.the.instrument.setup.procedure.be.performed.monthly.for.each.BD.FACSArray.bioanalyzer.used.to.analyze.a.BD.CBA.Flex.Set.experiment ..Run.the.setup.procedure.after.any.instrument.maintenance,.including.syringe.replacements,.regardless.of.time.since.last.setup .

Creating an Instrument Setup ExperimentIn.this.section,.use.the.BD.FACSArray.Experiment.Wizard.to.create.and.configure.an.instrument.setup.experiment.template ..

. 1 .. Start.the.BD.FACSArray.bioanalyzer.and.launch.BD.FACSArray.system.software.as.specified.in.the.BD FACSArray System Software User’s Guide..

. 2 .. Click.the.Experiment.Wizard.tool.( ).in.the.application.toolbar .

. . The.initial.view.of.the.Experiment.Creation.Wizard.is.displayed ..

. 3 .. Select.Default.and.click.Next .

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. 4 .. From.the.drop-down.menu,.select.BD.CBA.Flex.Set.and.click.Next .

Note:. In.subsequent.setups.you.can.select.the.most.recent.template.from.the.list .

. 5 .. Select.Yes.to.reserve.wells.to.check.instrument.settings.and.to.correct.for.optical.spillover ..Enable.NIR.and.Red.by.marking.their.checkboxes ..Click.Next ..

. 6 .. Select.the.default.settings.in.the.Enter the number of Calibrator Standard Replicates.view,.and.in.the.five.subsequent.views,.by.clicking.Next.six.times,.until.you.reach.the.Adjusting the plate layout.view ..

. 7 .. Select.Contiguous.Horizontal.and.click.Next .

. 8 .. Accept.the.default.settings.in.the.Loader.Settings.view.by.clicking.Next .

. 9 .. Accept.the.default.settings.in.the.Acquisition.Settings.view.by.clicking.Next .


. 10 .. Name.the.Experiment.BD CBA Flex Set Setup mmddyy.(in.place.of..mmddyy,.enter.today’s.date),.click.Next .

. 11 .. Click.Next.in.the.Saving.Wizard.Session.(you.do.not.need.to.save.the.wizard.session) ..The.Experiment.Creation.Wizard.advances.to.the.final.view ..

. 12 .. Review.the.wizard.selections.and.click.Finish ..The.specified.Experiment.is.created.and.it.opens.in.the.Prepare.workspace ..

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Preparation of Instrument Setup BeadsIn.this.section.the.instrument.setup.bead.samples.are.prepared.in.a.microwell.plate ..These.beads.will.be.used.to.establish.the.optimal.instrument.settings.for.the.Flex.Set.assays .

Note:. The.Experiment.Wizard.has.created.a.plate.template.with.12.wells.(the.minimum.number.of.wells.it.can.define) ..In.the.instrument.setup.Experiment,.only.5.wells.(A1.–.A5).are.used .

. 1 .. To.a.U-bottom.polystyrene.plate.(eg,.BD.Falcon™,.Cat ..No ..353910),.add.175.µL.of.Wash.Buffer.to.wells.A1,.A2,.A3,.A4,.and.A5 .

. 2 .. Add.25.µL.of.Instrument.Setup.Bead.F1.to.well.A1 .

. 3 .. Add.25.µL.of.Instrument.Setup.Bead.A1.to.well.A2 .

. 4 .. Add.25.µL.of.Instrument.Setup.Bead.F9.to.well.A3 .

. 5 .. Add.25.µL.of.Instrument.Setup.Bead.A9.to.well.A4 .

. 6 .. Add.25.µL.of.PE.Instrument.Setup.Bead.F1.to.well.A5 .

. 7 .. Shake.plate.using.a.digital.shaker.at.500.RPM.for.20.seconds ..

Adjusting Loader Settings

. 1 .. If.needed,.click.the.Prepare.tool.( ).in.the.application.toolbar.to.display.the.Prepare.workspace .

. 2 .. In.the.Plate.Editor,.click.well.A4.(see.figure) .

. 3 .. With.the.Inspector.window,.select.the.Acq.tab.and.adjust.the.loader.settings.as.follows.(see.figure) .

• Sample.Flow.Rate:.0 .5.µL/sec • Sample.Volume:.100.µL • Mixing.Volume:.60.µL • Mixing.Speed:.200.µL/sec • Number.of.Mixes:.1 • Wash.Volume:.200.µL

Note:.. Set.the.Sample.Flow.Rate.to.0 .5.µL/sec.to.provide.the.additional.time.needed.to.complete.adjustments.using.well.A4 .

. 4 .. In.the.Plate.Editor,.click.well.A5 .


. 5 .. With.the.Inspector.window,.select.the.Acq.tab.and.adjust.the.loader.settings.as.follows.(see.figure) .

. •. Sample.Flow.Rate:.0 .5.µL/sec

. •. Sample.Volume:.60.µL

. •. Mixing.Volume:.60.µL

. •. Mixing.Speed:.200.µL/sec

. •. Number.of.Mixes:.1

. •. Wash.Volume:.200.µL

Adjusting Scatter and Far Red Voltages

. 1 .. Click.the.Setup.tool.( ).in.the.application.toolbar.to.display.the.Setup.workspace .

. 2 .. Click.the.Parameters.tab.in.the.Instrument.window.to.display.the.voltage.settings.for.each.parameter .

. a .. Adjust.voltages.as.shown.in.the.figure .

. b .. Verify.that.Log.is.enabled.(checkbox.is.marked).for.each.parameter .


Adjusting Voltages with Well A4

In.this.section.the.plate.will.be.loaded.and.well.A4.will.be.acquired.to.set.the.singlet.gate.and.to.adjust.the.voltages.for.NIR-A.and.Red-A .

. 1 .. Place.the.microwell.plate.containing.the.setup.beads.in.the.loading.tray.of.the.BD.FACSArray™.bioanalyzer.so.that.well.A1.is.aligned.over.the.A1.position.label.on.the.plate.loader ..

. 2 .. With.the.Plate.editor,.click.Load.in.the.Plate.control.group .

. 3 .. In.the.Select.control.group,.click.None.to.deselect.the.wells,.then.click.well.A4.in.the.Plate.view ..

...

. 4 .. Change.the.Events.to.Display.to.50.evt .

. 5 .. Click.Setup.in.the.Acquisition.control.group ..Events.will.appear.in.the.Template.plots,.but.data.is.not.saved .

. 6 .. In.the.Template.window,.move.the.P1.gate.over.the.singlet.population.in.the.FSC-A.vs.SSC-A.dot.plot ..Resize.P1.if.necessary .

. •. Click.Setup.in.the.Acquisition.control.group.to.stop.acquisition .

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. 7 .. Adjust.the.threshold.settings.for.FSC.and/or.SSC.to.exclude.most.of.the.debris.without.cutting.into.the.bead.population .

. a .. Click.on.the.Threshold.tab.in.the.Instrument.window.to.display.the.threshold.settings .

. b .. Click.Add.to.add.SSC.threshold ..Ensure.the.And.radio.button.is.selected .

. c .. Click.inside.the.FSC.vs.SSC.dot.plot.where.the.beads.are.displayed ..This.will.allow.for.the.coordinates.of.the.cursor.inside.the.plot.to.be.displayed.in.the.lower-left.corner.of.the.plot .

. d .. Move.the.cursor.to.the.lower.left.edge.of.region.P1 ..Use.the.values.displayed.for.the.cursor.as.a.guideline.to.set.threshold.values ..The.x-coordinate.corresponds.to.the.FSC.channel.number.and.the.y-coordinate.corresponds.to.the.SSC.channel.number ..Use.caution.as.excluded.events.cannot.be.recovered.post-acquisition ..Typically,.both.FSC.and.SSC.thresholds.will.be.>.20,000,.but.in.some.instances.they.may.be.lower .

. 8 .. Right-click.in.the.statistics.box.and.select.Edit.Statistics.View .

. 9 .. Select.the.Populations.tab ..Mark.the.checkboxes.as.shown.in.the.figure.so.that.only.P1.#Events.and.P1.%Parent.are.enabled .


. 10 .. Select.the.Statistics.tab ..Mark.the.checkboxes.as.shown.in.the.figure.so.that.only.the.Yellow-A,.NIR-A,.and.Red-A.Median.and.the.Yellow-A.Mean.statistics.are.enabled ..Verify.that.Sort.by.Parameter.is.selected .

. 11 .. Click.OK ..The.Edit.Statistics.View.dialog.is.dismissed.and.the.Statistics.box.displays.the.selected.statistics .

. 12 .. Adjust.the.voltages.for.A4:

. a .. Click.the.Parameters.tab.of.the.Instrument.window,.if.needed .

. b .. Click.Setup.in.the.Acquisition.control.group.to.start.the.acquisition .

. c .. Adjust.the.NIR.voltage.setting.until.the.median.fluorescence.for.population.P1.is.the.NIR-A.channel.is.in.the.range.of.160,000.±.2,000 .

. d .. Adjust.the.Red.voltage.setting.until.the.median.fluorescence.for.population.P1.is.the.Red-A.channel.is.in.the.range.of.160,000.±.2,000 .

. e .. Click.Setup.in.the.Acquisition.control.group.to.stop.the.acquisition .

13.. If.you.run.out.of.sample.before.completing.the.adjustments:. a .. Click.Unload.in.the.Plate.control.group .. b .. Add.175.µL.of.wash.buffer.and.25.µL.of.Instrument.Setup.Bead.A9.to.

well.A4 ..Ensure.that.the.new.bead.sample.is.well.mixed .. c .. Click.Load.in.the.Plate.control.group .. d .. Click.None.in.the.Select.control.group.to.deselect.the.wells,.then.click.

well.A4.in.the.Plate.view .. e .. Click.Setup.in.the.Acquisition.control.group.to.start.setup.acquisition.of.

well.A4.and.repeat.step.12 .

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Adjusting Voltages with Well A5

In.this.section.well.A5.will.be.acquired.to.adjust.the.voltage.for.the.Yellow.detector .

. 1 .. With.the.Plate.Editor:

. a .. In.the.Select.control.group,.click.None.to.deselect.the.wells .

. b .. Click.well.A5.in.the.Plate.View .

. 2 .. Click.on.the.histogram.plot.parameter.label.and.use.the.drop-down.menu.to.change.the.histogram.parameter.to.Yellow-A.(see.Figure A) .

. 3 .. Click.Setup.in.the.Acquisition.control.group.to.start.acquisition.of.well.A5 .

. 4 .. As.the.sample.is.being.acquired,.adjust.the.Yellow.voltage:

. a .. Adjust.the.voltage.setting.for.Yellow.until.the.Mean.fluorescence.for.population.P1.in.the.Yellow-A.channel.is.60.+.5.(see.Figure B) .

. b .. Click.Setup.in.the.Acquisition.control.group.to.stop.acquisition..(see.Figure C) .

. .

Figure A

Figure B

.

Figure C

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Correcting for Optical Spillover

In.this.section,.wells.A1,.A2,.and.A3.will.be.acquired.to.provide.data.for.calculating.optical.spillover.corrections ..After.the.spillover.calculation,.well.A5.will.be.acquired.to.complete.the.setup.Experiment .

. 1 .. In.the.Select.control.group,.click.None.to.deselect.the.wells ..In.the.Plate.view.click.wells.A1,.A2,.and.A3 .

. 2 .. Click.Acquire.in.the.Acquisition.control.group.to.begin.acquisition.of.the.optical.spillover.wells ..

. 3 .. If.a.gating.error.dialog.is.displayed,.click.OK ...

. 4 .. Click.the.Analyze.tool.( ).in.the.application.toolbar.to.display.the.Analyze.workspace .

. 5 .. With.the.Plate.Editor,.click.well.A1 .

. 6 .. In.the.Template.window,.adjust.the.singlet.and.histogram.gates.as.necessary.to.encompass.populations.as.shown.in.the.figure .

. 7 .. Click.well.A2 ..Adjust.singlet.and.histogram.gates.as.necessary.to.encompass.populations,.as.shown.in.the.figure ..

. 8 .. Click.well.A3 ..Adjust.singlet.and.histogram.gates.as.necessary.to.encompass.populations,.as.shown.in.the.figure ..


. 9 .. Select.Instrument.>.Calculate.Spillover ..

. . When.the.spillover.calculation.has.completed,.the.Spillover.dialog.is.displayed ..

. 10 .. Click.OK .

. 11 .. Click.the.Setup.tool.( ).in.the.application.toolbar.to.display.the.Setup.workspace .

. 12 .. With.the.Plate.Editor:


. b .. Click.well.A5.in.the.Plate.view .

. c .. Click.Acquire.in.the.Acquisition.control.group .

. d .. When.acquisition.is.completed,.click.Unload.in.the.Plate.control.group,.and.unload.the.setup.plate.

Saving the Instrument Setup Experiment as a TemplateIn.this.section,.a.BD.FACSArray™.setup.Experiment.template.is.created.and.saved ..To.complete.the.setup.Experiment.configuration,.the.experiment.must.be.closed.and.reopened .

Note:. If.an.Experiment.template.is.saved.without.first.closing.and.reopening.the.Experiment,.it.is.possible.that.the.stopping.gate.criteria.will.not.be.properly.recorded.in.the.template.file ..

. 1 .. Click.the.Prepare.tool.( ).in.the.application.toolbar.to.display.the.Prepare.workspace .


. 2 .. Double-click.the.icon.of.the.currently.open.setup.Experiment.in.the.Browser.to.close.the.Experiment ..

. 3 .. Double-click.the.icon.of.the.now.closed.setup.Experiment.in.the.Browser.to.reopen.the.Experiment ..

. 4 .. Select.Experiment.>.Save.As.Template.from.the.menu.bar ..

. 5 .. In.the.Save.Experiment.as.Template.dialog.name.the.template.BD CBA Flex Set Setup mmddyy.(in.place.of.mmddyy,.enter.today’s.date),.and.click.OK .

. . Instrument.setup.is.now.complete ..If.desired,.the.BD.FACSArray™.system.software.can.be.closed .


Transferring the Experiment TemplateAfter.a.modified.BD™.CBA.Flex.Set.Experiment.template.has.been.saved,.it.should.be.made.available.to.FCAP.Array™.software.by.copying.the.template.to.the.FCAP.Array.software.Templates.folder ..FCAP.Array.software.uses.the.template.when.it.exports.an.Experiment.back.to.BD.FACSArray.system.software.(see.Exporting the Experiment File on.page.21) .

Set Up Instrument

TransferTemplate

ExportExperiment

ImportExperiment

AquireData

AnalyzeData


. 1 .. Copy.the.modified.BD.CBA.Flex.Set.template. .xml.file:

. a .. Using.the.folder.system.of.your.BD.FACSArray.workstation,.open.the.folder:.C:\ Program Files\ BD FACSArray System Software\ templates\ BD CBA Flex Set Setup mmddyy (depending.on.the.configuration,.this.location.may.be.on.the.D:\.drive) .

. b .. Copy.the.file.BD.CBA.Flex.Set.Setup.mmddyy .xml.by.right.clicking.the.file.icon.and.selecting.Copy.from.the.context-sensitive.menu ..Close.the.folder .

. 2 .. Paste.the. .xml.template.file.to.an.appropriate.location.in.the.computer.containing.FCAP.Array.software:

. a .. Open.the.folder:.C:\Program Files\ Soft Flow\ FCAP Array Software v1.0\ Templates.(depending.on.the.configuration,.this.location.may.be.on.the.D:\.drive) .

. b .. Paste.the.template. .xml.file.by.right-clicking.in.the.file.list.and.selecting.Paste.from.the.context-sensitive.menu ..Close.the.folder .


Routine BD™ CBA Flex Set Data Acquisition and AnalysisThe.daily.routine.for.acquiring.and.analyzing.BD™.CBA.Flex.Set.assays.with.the.BD.FACSArray™.should.begin.with.the.creation.of.an.Experiment.in.the.FCAP.Array™.software ..This.FCAP.Array.Experiment.will.contain.all.of.the.information.regarding.the.number.of.standards.and.samples.to.be.acquired.for.the.experiment ..Once.the.FCAP.Array.Experiment.is.created,.it.is.then.exported.to.the.BD.FACSArray.software.to.be.used.for.sample.acquisition ..Once.the.Flex.Set.assay.data.files.are.acquired,.the.data.will.be.analyzed.in.the.FCAP.Array.software ..Refer.to.the.FCAP Array Software User’s Guide.for.more.detailed.information.about.the.features.of.the.software ..

Creating an Experiment Using the FCAP Array™ SoftwareAn.FCAP.Array.software.Experiment.must.be.created.before.sample.data.can.be.acquired .

. 1 .. Launch.the.FCAP.Array.software .

. 2 .. Enter.your.user.name.and.password.and.click.Log in ..

. . The.default.user.login.is.Administrator.and.password.is.welcome ..

Using the Experiment WizardIn.this.section.the.Experiment.Wizard.is.used.to.create.an.Experiment.document .

. 1 .. Do.one.of.the.following.to.start.an.Experiment.Wizard.session .

• If.the.Quick.Start.dialog.is.displayed,.click.the.New Experiment.icon ..

• Otherwise,.click.the.New.Experiment.icon.in.the.application.toolbar .


Entering Test Samples and DilutionsIn.the.Test.Samples.and.the.Dilutions.and.Replicates.views,.enter.information.about.your.test.samples .. 1 .. Click.Next.to.advance.to.the.Test.Samples.view.and.specify.the.number.of.

test.samples ... 2 .. Change.the.test.sample.names,.if.desired .. a .. Click.the.first.sample.to.highlight.it .. b .. Click.again.on.the.first.test.sample.name.to.obtain.an.editable.field .. c .. Edit.the.first.test.sample.name.and.press.Enter.to.advance.to.the.next.

line.and.obtain.an.editable.field .. d .. Edit.the.remaining.test.sample.names .. 3 .. Click.Next.to.advance.to.the.Dilutions.and.Replicates.view .

Note:. The.default.dilution.factor.is.1 .00.(no.dilution) .

. 4 .. Change.the.dilution.factor.for.any.test.samples.where.it.is.appropriate ..

Creating a New Plex. 1 .. Click.Next.to.advance.to.the.Selecting.Saved.Plex.view .

. 2 .. Verify.that.**New.Plex**.is.selected.in.the.browser.window ..Previously.saved.plexes.can.be.selected.by.expanding.the.All.Saved.Plexes.folder .

. 3 .. Click.Next.to.advance.to.the.Plex.Components.view .

Choosing Plex ComponentsSpecify.the.Beads.needed.for.the.Plex ..If.the.bead.list.is.empty.manually.enter.bead.information.by.clicking.the.Edit.button.or.visit.bdbiosciences.com/flexset to.download.the.master.bead.list ..In.the.Plex.Components.view.click.Edit.to.access.the.Bead.Library.and.to.import.the.bead.list ..Refer.to.the.FCAP Array Software User’s Guide.for.more.information.about.managing.the.Bead.Library ..

. 1 .. Add.the.Beads.to.be.collected.in.the.Experiment.by.selecting.the.bead.

from.the.left.column.then.using.the.( ).button.to.add.the.bead.to.the.Selected.beads.list.on.the.right ..

Note:. Because.this.FCAP.Array.software.Experiment.is.being.created.prior.to.data.acquisition.(design.first.workflow),.data.is.not.available.for.completing.the.Clustering.Parameters.view ..Therefore,.skip.both.the.Clustering.Parameters.view,.and.the.associated.Analyte.Assignment.view.for.now,.and.return.to.these.activities.after.acquisition.(refer.to.the.FCAP Array Software User’s Guide.Chapter.7;.Data.Analysis) ..


Specifying StandardsIn.this.section,.specify.the.fitting.equation.and.standards.for.a.quantitative.assay .

. 1 .. Click.Next.twice.to.advance.to.the.Qualitative/Quantitative.view .

. 2 .. Verify.that.Quantitative.is.selected.and.Uniform fitting equations.is.enabled ..

. 3 .. Click.the.table.cell.in.the.top.row.under.Fitting equation.to.activate.its.pull-down.menu.and.select.the.desired.curve-fitting.model ..The.4-.and.5-.parameter.logistic.models.are.most.commonly.used ..

. 4 .. Click.Next.to.advance.to.the.Standards.view ..

. . In.the.Standards.view.for.a.Quantitative.assay.the.number.of.standards,.the.standard.concentrations,.and.the.number.of.replicates.for.each.standard.are.designated ..

. 5 .. Specify.the.appropriate.number.of.standards.in.the.experiment ..The.assay.procedure.detailed.in.the.BD™.CBA.Flex.Set.Master.Buffer.Kit.manuals.utilizes.10.standard.curve.points .

6.. Click.the.table.cell.of.the.Unit.row.and.CC.column.to.specify.the.appropriate.concentration.units.from.the.drop-down.menu ..

. 7 .. In.the.CC.column,.enter.the.concentration.values.that.correspond.to.each.standard.point ..It.is.recommended.to.run.the.zero.standard.first.followed.by.the.standard.dilutions.from.least.concentrated.to.most.concentrated .

Creating a Plate LayoutIn.this.section,.specify.the.sample.arrangement.on.the.plate ..BD.FACSArray™.system.software.acquires.data.according.to.the.plate.layout.specified.in.FCAP.Array™.software ..Use.this.section.to.generate.a.template.that.can.be.printed.to.ensure.the.plate.is.loaded.correctly .

. 1 .. Click.Next.three.times.(once.on.each.of.the.three.consecutive.views).to.advance.to.the.Plate.Layout.Options.view ..

. 2 .. Verify.that.96 well plate.is.selected ..Note:. The.BD.FACSArray.bioanalyzer.is.not.compatible.with.any.other.

plate.or.rack.format ..

. 3 .. Choose.an.arrangement.method.that.matches.how.the.test.samples.are.going.to.be.laid.out.in.the.plate ..

. 4 .. Select.the.Place samples at the end.check.box.to.designate.the.first.wells.as.the.standards .

. 5 .. Click.Next.to.advance.to.the.Experiment.Name.view .

. 6 .. Enter.an.appropriate.name.for.the.Experiment.name .

. 7 .. Click.Finish.to.close.the.Experiment.view .

. . The.new.Experiment.opens.in.the.main.window.in.the.Plate.Layout.view,.and.a.Save.As.dialog.is.provided.to.save.the.Experiment .

. 8 .. Create.a.new.folder.on.the.desktop.called.FCAP.Assays ..Navigate.to.the.FCAP.Assays.folder .Note:. The.experiment.file.can.be.saved.to.any.location .

. 9 .. Click.Save .

Unless otherwise specified, all products are for Research Use Only. Not for use in diagnostic or therapeutic procedures. Not for resale. 21US Orders: 877.232.8995

Exporting the Experiment File

Set Up Instrument

TransferTemplate

ExportExperiment

ImportExperiment

AquireData

AnalyzeData


. 1 .. If.needed,.click.the.Plate.Layout.tab.to.display.the.Plate.Layout.view .

. 2 .. Click.Export.Experiment ..The.Experiment.Export.Wizard.opens.in.the.Select.Template.view ..

. 3 .. Select.the.BD.CBA.Flex.Set.Setup.mmddyy .xml.template.that.was.created.in.Saving the Instrument Setup Experiment as a Template.on.page.15 ..

. 4 .. Click.Next ..The.wizard.advances.to.the.Set.Stopping.Rule.view .

. 5 .. Set.the.number.of.events.per.Bead.to.be.acquired ..The.recommended.value.is.300.events.per.Bead ..

Note:. The.total.number.of.events.is.determined.by.the.software.by.multiplying.the.events.per.Bead.by.the.number.of.Beads.in.the.Experiment ..

. 6 .. Click.Next ..The.wizard.advances.to.the.Select.Folder.view .

. 7 .. Click.Browse ..A.Browse.for.Folder.dialog.is.displayed .

. 8 .. Navigate.to.C:\Program.Files\BD.FACSArray.System.Software.and.create.a.new.folder.there.named.FCAP Array Files xml (this.location.may.be.on.the.D:\.drive.on.some.computers) .Note: The.exported.experiment.file.can.be.saved.to.any.location .


. 9 .. Click.OK .

. 10 .. Click.Finish ..The.wizard.advances.to.the.Complete.view ..

. 11 .. Click.Close ..A.new.folder.is.created.in.the.FCAP.Array™.files.folder.to.hold.the.exported.XML.Experiment.file ..The.New.folder.name.is.the.name.of.the.FCAP.Array.Software.Experiment ..

. 12 .. Exit.from.FCAP.Array.software .

Importing Experiment File into BD FACSArray™ System Software

Set Up Instrument

TransferTemplate

ExportExperiment

ImportExperiment

AquireData

AnalyzeData


. 1 .. Start.up.the.BD.FACSArray™.bioanalyzer .

. 2 .. Launch.BD.FACSArray.system.software .

. 3 .. In.BD.FACSArray.system.software,.select.File>Import.Experiments .

. 4 .. Navigate.to.the.folder.that.was.created.in.step.8.in.the.previous.section .

. . Do not open the folder..Select.it,.and.click.Import ..

. . The.Imported.Experiment.is.added.to.the.BD.FACSArray.database.and.appears.in.the.Browser ..

. 5 .. Locate.the.imported.Experiment.in.the.Browser.of.the.BD.FACSArray.software,.and.double-click.the.Experiment.icon.to.open.the.Experiment ..

Acquiring DataBefore.acquiring.data,.a.plate.must.be.prepared.with.the.Flex.Set.assay.samples ..For.detailed.information.on.preparing.assay.samples.for.a.plate,.refer.to.the.appropriate.BD™.CBA.Flex.Set.Master.Buffer.Kit.manual .

. 1 .. Ensure.that.the.sample.layout.of.the.BD.FACSArray.Experiment.reflects.the.layout.in.the.prepared.plate.of.BD.CBA.Flex.Set.assays.about.to.be.acquired ..


. 2 .. Click.the.Setup.tool.( ).in.the.application.toolbar.to.display.the.Setup.workspace ..

. 3 .. Place.the.assay.plate.(containing.the.standards.and.test.samples).in.the.loading.tray.of.the.BD.FACSArray.bioanalyzer.so.that.A1.is.aligned.over.the.A1.position.label.on.the.plate.holder ..

. 4 .. Click.Load.to.load.the.prepared.plate.and.change.Events.to.Display.to.5000 .

. 5 .. Verify.that.the.correct.number.of.bead.clusters.is.being.displayed.and.identified.before.the.entire.plate.is.acquired .


. b .. Click.on.well.A1.in.the.Plate.view .

. c .. Click.Acquire.in.the.Acquisition.control.group.to.start.acquisition.of.well.A1 .

. d .. After.well.A1.is.collected,.inspect.the.plot.to.make.sure.that.the.correct.number.of.bead.clusters.are.being.displayed ..Make.any.adjustments.to.the.singlet.gate.or.thresholds.if.necessary .

Note:. The.data.file.can.be.exported.as.FCS2 .0.and.investigated.in.FCAP.Array.software.by.going.to.the.Tools.menu.>.Clustering.Test.and.reviewing.the.data.file.within.this.tool.to.ensure.that.the.proper.number.of.bead.clusters.is.recognized ..Refer.to.the.FCAP Array Software User’s Guide.for.more.information.on.this.tool ..

. e .. After.the.bead.populations.have.been.verified,.click.All.in.the.Select.control.group.to.select.the.entire.plate.for.acquisition .

. 6 .. Click.Acquire.to.start.acquisition.of.the.wells .

. . A.warning.will.appear.asking.if.you.want.to.overwrite.well.A1,.click.OK .

. 7 .. When.acquisition.completes,.click.Unload .

. 8 .. Click.the.Prepare.tool.( ).in.the.application.toolbar.to.display.the.Prepare.workspace .


. 9 .. In.the.Browser,.select.the.Experiment.that.was.just.acquired .

. 10 .. Export.the.FCS.2 .0.list-mode.files.from.the.this.Experiment:

. a .. Select.File.>.Export.>.FCS

. b .. Select.FCS.2 .0.as.the.file.version.to.export .

. c .. Click.OK ..

. d .. Click.Save.to.save.the.FCS.files.to.the.default.location.D:\BD.Export\FCS .

. 11 .. Shut.down.the.BD.FACSArray™.bioanalyzer .

See.the.shutdown.procedure.in.the.BD FACSArray System User’s Guide ..

Analysis of Sample DataFollow.the.instructions.in.Chapter.7:.Data.Analysis.of.the.FCAP Array User’s Guide.in.order.to.analyze.the.sample.data.just.acquired.on.a.BD.FACSArray.bioanalyzer .

Troubleshooting Tips

Problem

Increase the threshold or enable dual FSC and SSC thresholds. Position the cursor on the lower boundaries of the singlet population on the FSC/SSC plot and use the coordinates as a guideline to set threshold values. Use caution as excluded events cannot be recovered post-acquisition.

Suggested Solution

Debris (FSC/SSC) during sample acquisition

Bead clusters merging or incorrect number of bead clusters

Low event count

Clogged filter plate

Be sure that setup beads are at the appropriate MFI targets as directed in the instrument setup procedure and repeat monthly instrument setup procedure if necessary. Run daily instrument QC to ensure that instrument is performing well prior to performing the assay. Instrument service may be required.

The beads can precipitate, thoroughly vortex individual capture bead bulk vials prior to preparation of master bead mix and vortex the master bead mix prior to dispensing into the individual sample wells. Shake plate thoroughly prior to acquisition.

Serum and plasma proteins can settle and clog the membrane during incubation. Dilute samples further or perform assay incubations in a standard polystyrene U-bottom plate (BD 353910) and transfer to the filter plate immediately prior to aspiration. Resuspend the beads well prior to transfer.

Ensure that the stopping rule, singlet gate, and thresholds are set correctly.

Check loader settings. Default settings are in the BD CBA Flex Set wizard template.

Ensure vacuum is not too strong and filter membranes are not compromised.

Notes

Notes

BD Biosciences10�� Torreyana RoadSan Diego, CA �2121Customer/Technical ServiceTel ��.2�2.�� (US)Fax �00.�2�.�6��bdbiosciences.com

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