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SonyMA900CellSorter

70and100µmchipsarecurrentlyinuse

130chipsarebeingupdatedreleaseplanned2020

OnFirstuseroftheDayStartUpProcedure:IftheInstrumentisonandcalibratedskiptopage71. Turn on the air pressure (counterclockwise) to the instrument, this must be done first. The blue valve should be

inline as pictured above. It is clearly marked and located on the left back wall area of the instrument

2. FirstcleanthesampleandsortchamberswithCavicide.Donotsprayinsidethechamberasitmaydirtythewindowopticsthatmonitorthesidestreams.Soakakimwipeandthencleanallthesurfacesinsidethechamberexceptthesortplates.Thesortplatescanonlybecleanedwithcleanwateror70%ethanol.Tocleantheplatescarefullyremoveoneplatebyundoingthethumbscrews.Leavethesortplateinsidethesortchambertopreventaccidentallydroppingit.Workinginsidethesortchamber,wipebothplatesinonedirectionwithalcoholswipefromtoptobottom.Cleanthebaseofthesortchamber,pointoutfloordrainareatoclean.

3. Fillsheathtanktobelowtheupperweldline.Checkthepressurereleaseringontopofthetanktobesureitisdowninthegrooveonthetopofreliefvalve.

4. Emptythewastetank,addbleachtomaintain10%forbiosafetydecontamination.Holdthemetalfittingsinthecapstableandturntheoutercaponlytoremove.Makesurethefilteronthewastecappointsupandisnotallowedtogetwet.Thereisabeakeronthefloortoplaceitintokeepitorientedinthecorrectposition.

5. Staffgenerallyfillsthethewater,70%ethanoland10%bleachtanklevels.Usersneedthesheathfullandwasteemptytorun.

6. PoweruptheMA900.

FACSCoreLab

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7. TheAerosolManagementSystemisuniqueforthissystem.Itisonlyactivatedtoclearaerosolsfor30sec-1minutebeforeopeningthecollectionchamber.TheunitisNOTONduringthesort.ONLYactivatedbrieflybeforeopeningthesortchamber.Demonstrateon/offfunctionsoftheBuffalounitanduseofthefootpedalcontrols.

8. *Oncethesystemhasbeensetup,inordertoemptythewasteorfillsheathyoumustputtheMA900in“standby”mode.Directionsforputtingthemachineinstandbyarelocatedonpage6*.Ifyoufailtodothisitwillresultinhavingtorecalibratethesystemandasignificantlossoftime.

9. LogintoWindows.Account:guest1,password:guest.10. OncetheMA900isinStandby,starttheCellSorterSoftware.11. Loginwithyourusernameandpassword.

AutomatedAlignmentandSortSetup(30minutes)ReadallpromptsandrespondATTENTION:QCtakes30-40minutes,tohelpassistinpassingcleantheplates,andaspirator.Duringthefluidicsstartupselectcleansampleline,andsheathfilterde-bubblepriortheQCisrecommended.

o Sidestreamsmaynotformiftheplatesarenotcleananddryo IfsamplelineisdirtyitmaynotdeliveradequatebeadstopassQCo Airinsheathfilterwillcauseinstabilityofthestreamandcausefailureofsortparameterso Fullsheathtankpromotesstability

1. Afterlogin,youarepromptedtoscanandloadthenewchip–Chipsaregoodfor24handthepreviousday’schipcanbereusedif<24hhaspassed.Ifcontinuinguseonthecurrentchip,simplyopentheinstrumentfrontandreloadthecurrentchiponceitisejected.Load/exchangethechipfollowingtheonscreeninstructions.Writedateandtimeonthepackageifusinganewchip.i. Toloadachip–feedthechipuntilthesoftstop,thengentlyengagethelast¾inchdemonstrate.ii. Selectthedesiredlasers,the488nmlaserismandatoryselectotherdesiredlasers.iii. SelecttheStandardfilterconfigurationfortheinstrument(thisisthesystemnotyourapplicationsetting).iv. Followpromptsonscreentochecksamplelineforbackflush.2. DuringthefluidicsstartupselecttheSheathFilterDe-bubbleoptionandSamplelinecleaning(thiswilldoa

10%BleachandSterileDiH20cleanfor8-10minutes)availableonthebottomofthescreenduringstartupbeforerunningtheQCbeads.

3. Beforeyourunthebeadschecktobesuretheplatesarecleananddry.OccasionallytheywillgetwetduringthefluidicsstartupandiftheyarenotcleanedanddryitmaycauseafailureinyourQCandasignificantlossoftime.

4. RunAutocalibration.NOCAPontube.i. MixtheSonybeadsvialgentlyanddispenseabout1mlintoatube(canbepolystyreneorpolypropylene),place1mlofSonybeadsinthecorrect5mltubeholderonmachine.Whenprompted,loadthecalibrationbeads.Makesurethetubegoestothebottomofthetubeholder.

ii. SelectedtheTargetedSteamoption.iii. Steponeofthecalibrationalignsthechipandfindsthe

targetfluorescentsensitivityandsetsthelaserdelay.Thistakesapproximately10minutes.Steps2-4finetunethestreamprofile,sidestreams,dropchargeandsortdelay.Thistakesapproximately30minutes.

iv. Ifautocalibrationfails,checkthelogfileforfailureexplanation,checkthattheplatesarecleananddry,doasheathfilterde-bubble.

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v. Ifthisdoesnotresolvetheissue,checktheopticalfilters,sheathtankseal,checkfrontcytometerpaneforliquidlevels.Textorcallstaffforassistance.

ExperimentSetup1. Filterallsamplesincludingcontrols40u

mesh.2. Selecttheexperimentyouwouldliketo

use,ortheBlankTemplate.3. Ontheupperright,changetheexperiment

name.4. Fillinotherinformationforyour

experiment.5. Checkoruncheckthefluorescent

parametersthatareneededandnametheparametersifyouwish.

6. Turnon/offlasersforyourexperiment.Besurethatthe488nmlaserison.

7. ClickCreateNewExperimentinthelowerrightofthemonitor.

Whenrunningacompensationmatrix1. Thereare2methodsforcompensationavailablethe

wizardandmanual.2. Choosetostartcompensationwizardifyouaresorting

withmultiplefluorochromesandhavepreparedsinglecolorcontrols.

3. ClickOKtocontinue4. Flowrateiscontrolledbysamplepressuresetting.5. Followthewizard’sinstructionsandrunthenegative

controlandadjusttheFSCandBSCinDetector&Thresholdsettingstogetthepopulationsonscale.Brieflyloadafullystainedsampleatthispoint(donotrecordit)andverifythatnoneofthepositivepopulationsareoffscale.Iftheyare,lowerthePMTvaluetogetthembackonscale.

6. Thenreloadthenegativecontrolandrecord.Adjustthegatesuchthatitisaroundthecorrectscatterpopulation.

7. Acquirethesingle-stainedcontrols,adjustthegatearoundthepositivepopulations,thenclickCalculateMatrix.Theshapeoftherecordbuttonchangesfromcirculartosquarewhendataisbeingrecorded.

8. ClickFinishtoendtheWizard.

o Ifnocompensationisnecessary,selectDetector&ThresholdSettings.o AdjustFSCgainandBSCgainsuchthatyourcellsareonscale.

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o Acquireenougheventsforthesampletobesortedsothatyoucansetgates.Pausetheacquisition.o Createplotsontheworksheet.Doubleclickinsidegatestocreategatedplotsorchangethegatesby

clickingonthegatenameatthetopofaplot.BesuretoincludeasingletgatebasedonFSC-AvsFSC-HorFSC-HvsFSC-W

o Adjustgatesettingsforpopulationsofinterest.o Setthevaluetorecordto5,000or10,000events.Ifthepopulationisrare,recordmoreevents.

9. Startmakinggraphsandapproximategatesforyourexperiments.Createdelements,plotsandapproximategatesbeforecollectingdatawillallowthemtobeavailableinallthetubes.Ifyouforgetyoucancopytheworksheetelementstothefollowingtubes.

Tubesortingsetup1. Selectthedesiredsortmode.PurityorSemi-Purityisrecommended.

2. Setthesortgates.Indicatethenumberofcellstobesortedorleavethevalueat0tosortcontinuously.

3. Tochecktrajectoryofyoursidestreams,loadatargetingtubewithtapeorparafilmontopsoyoucanseethe

dropdepositedinyoursamplecollectionholder.Placesamplecollectionholderinsortcollectionchamber.4. Inthecytometerribbonselecttheblacktoolboxlabelledsettings.SelectAdvancedSettingstabnearthe

bottom,inthestreamwindowbottomleft,selectLoadCollection,thenhitStarttodepositfluidandcheckstreamtrajectory,adjustifnecessary,byclickingthearrowsontheright.Oncetargetsaredefinedloadcollectiontubeswithmediaforsort.

5. Placethecollectiontubeholderintothecollectionareaandsetcollectiontubes.6. Click“NextTube”tocreateanewtube.7. Click“LoadCollection”.8. Makesureflowrateisstable,click“Start”toloadsampletubeandstartacquiring.9. Hit“Sort&RecordStart”tosortandsavedataforthesample.10. Runthesamplepressureatamaximumof6duringsorting.11. FromtheCytometertab,youcanadjusttemperaturecontrolforthesampleandcollection,chamberlights

andagitation.12. Monitorthecollectiontubeschangethemwhentheyarefull.TurnonAerosolmanagementunitfor60

secondsbeforeopeningthechamber.Theunitmustbeoffforsorting.

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13. Tochangetubes:Pausethesample,UnloadCollection,switchtubes,loadcollectionandthesortwillcontinue.14. Tostopthesort,presstheblackStopbutton.On/offaerosolmanagement.TheAerosolManagementSystem

onthisunititisONLYonwhenyouareremovingasampleaftersorting,ortocleantheunitafteraclog.Turnontheunitusingthepowerbuttonbottomleftfront.Hitthefootpedalonthefloortoactivatesuction.Leavetheunitevacuatingfor30-60seconds.THEUNITISOFFWHENYOUARESETTINGUPANDSORTING.ItisonlyactivewhenthedooristobeopenedafterBSL-2sortingorineventofaclog.Turnoffunitimmediately.

SortingintoaPlate1. Installsplashguard,platestandandplateholder(storedinrefrigerator).2. ChangetheSortMethodto96or384-wellplate.3. Startthesample,thenPausewhenenoughcellsareseentosetsortinggates.Pausethesampleandsetthe

gates.4. ClickonSortSettingstoopenthe96wellsortsetupandplateadjustments5. Loadaplatewiththecoveron.6. SelectthePlateAdjustmenttab.7. Chooseeithertodeflectemptydropsofsheathfluid,ortorunthesample(preferred).8. Select‘4cornersandcenterwell’9. Ifrunningthesample,selectthegatetouse(typicallythesinglets).10. Click‘Start’andtheMA900willsort50dropsintothecornerwellsandacenterwell.11. Whenitfinishes,PausethesampleandUnloadtheplate

(automaticifthesampleisnotrunning).Youcanremovetheplatetocheckthedroppositionsoverthewells.Notetheadjustmentsneeded,andre-loadtheplate.Clickoneachcorneroneatatime,andadjustthedropposition.Thedefaultadjustmentof1mmcanbechangeddownto0.1mmifdesired.Oncetheadjustmentsarefinished,click‘Start’again.

12. Removetheplatetobesurethattheadjustmentsarecorrect,re-doifnecessary.

13. Selectthewellstobesorted:o InthePlateSortSettingstab,highlightthewellstobesorted.SelectthegateandStopCount,thenclick

Add.o ChecktheIndexSortboxifdesired.

14. Removetheplatelid.15. Click‘IndexSort&RecordStart’.16. Whensortisfinished,youwillhavetheoptiontocontinuesortinganadditionalplate.Clickcontinueifyou

wishtosortadditionalplateswiththesamesample.Youwillhavetheopportunitytochangesortcriteria.Selecting“no”willrequirethatyouunload/reloadthesample.On/offaerosolmanagement

17. Removetheplateholder(storeinrefrigerator)andsplashguard.18. Toanalyzeindexsortdata,clickontheWorksheetToolsand‘AnalyzeIndexData’

*Ifthewasteneedstobeemptiedorsheathfilledaftertheautocalibrationhasalreadybeendone,itwillneedtobeputinstandbymode.Ifyoudonotputthemachineinstandbymodeandattempttoemptythewaste,thestreamwillshutoffandyouwillhavetoruntheautocalibrationagain.Toputthemachineinstandbymode:

1. Fromthecytometerribbon,select“settings”(a)2. Onthesettingspanel,select“advancedsettings”

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3. Onthe“pressureoptions”tab,select“standby”(b)4. Onceyouhavereplacedthetankafteremptyingandaddingbleach,orfilledthesheathtankselect“ready”(c)

DataExport1. FCSfileexport:rightclickontheExperiment(scrolluppastallthesampletubestothetopofthe

experiment),andselectExportasFCSFiles,orintheExperimenttab,selectExportFCSFilesfromtheribbon.

2. The…buttonletsyoubrowsetotheappropriatefolder.1. clickexportàclickclose

3. APDFoftheexperimentlayout,includingsortsetupandascreenshotofthegatescanbesavedbyclickingcustomprint(intheWorksheetToolsRibbon).

4. Experimentexport:FromtheFilemenu,selectDatabase.Clickontheexperimenttoexport,thenonthearrowstomoveittotheexportwindowontheright.

5. The…buttonletsyoubrowsetotheappropriatefolderC:\Facsdata.6. Thissavestheentireexperimentandallowsyoutosavethedata,theplotsandthegates.Itisrecommended

thatyoudothisexportinordertohaveafullbackupoftheexperiment.Itcanthenbedeletedandimportedbackintothedatabaseifnecessary.

7. Onthedesktopusethedatauploadicontouploadyourexperimenttobox,thesameprocedureforallourcytometersisused.

8. Checktheschedulertoseeifyouarethelastuser

CleanupIfNOT,thelastuseroftheday:1. FromtheCytometertab,selectBleachClean.

Prepare15mltubewith10mlof10%bleach.Placeontubeholderandproceedwithcleaning.Thiswilltake6minutes,andapproximately7mlofbleachwillberunthroughthesampletubingandchip.

2. FromtheCytometertab,selectShutdownRinse.Preparea15mltubewith12mlofDiwater.Placethetubeontheholderandproceedwiththerinse.Thiswilltakeanother6minutes.

3. Logoutofthesoftware.4. Whentheboxcomesupaskingifyouaresureyou

wanttologout,checktheboxthatsays“Keepsortcalibration.”Ifyoudonotcheckthebox,thenextpersonwillhavetoruntheautocalibrationagainbeforesorting.

5. Ifthereismorethana2hourgapbetweenlog-ins,themachinewillshutthestreamoffandtheautocalibrationwillhavetoberunagainatthenextlog.

ShutDown1. Ifthelastuser,fromtheCytometertab(a),selectHardwareandSoftwareShutdown(b).Thecleaning

wizardwillstart.Preparea15mltubewith12mlof10%bleach.Placeontubeholderandproceedwithcleaning.Thiswilltake6minutes,andapproximately7mlofbleachwillberunthroughthesampletubingandchip.

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2. Afterthebleachcleaningisfinished,youwillbepromptedtopreparea15mltubewith12mlofDiwater.Placethetubeontheholderandproceedwiththerinse.Thiswilltakeanother6minutes.

3. SelectShutdowntopowerdowntheinstrumentandyouwillalsobeloggedoutofthesoftware.

4. Turnofftheairpressurewithswitchonwall(Rotatevalvetohorizontalpositionnotinline

withthefitting).

Off

QuickStartInstructionsifthemachineisalreadysetup.Whentheprevioususerhasalreadysetupthechipandlefttheinstrumentonforyou.Whenthelastuserloggedouttheymusthaveclickedon“Keepautocalibrationfornextuser”.Themachineisalreadycalibratedforyou.1.Logintothesoftwarewithyourusernameandpassword.Createyourexperimentorselectatemplateyouhavesaved.Selectdesiredlasersandparametersandlabelfluorophores.

2.IftheWasteandSheathfluidlevelsarelow(checktheinstrumentpanel),itisrecommendedthatyourefillthemnow.Todoso,youshouldfirstputtheinstrumentin“Standby”mode.Thisturnsoffthestreamanddepressurizestheinstrument.(Cytometertab>Settings>“Advancedsetting”>“PressureOptions”>“Standby”button).Donotclosethisdialogbox.

3.INSTANDBYMODE:Emptythewastecontainerandrefillthesheathfluid.Besuretodepressurizethesheathfluidtankbydisconnectingtheairpressurehose.Ithasasnap-fitconnection.Forthewastecontainer,holdthefittingstationaryandtwisttheplasticwhitecap.Donottiltthewastecontainerfitting.Keepthefilterpointedsoitdoesnotgetwet.Putitinsidethebeakertotheside.Thispreventsliquidfromenteringandcloggingtheairfilter.

4.INSTANDBYMODE:CleanthesampleandsortchamberswithCavicide.Thesortplatescanonlybecleanedwithsterilewateror70%ethanol.Tocleantheplatescarefullyremoveoneplatebyundoingthethumbscrews.Leavethesortplateinsidethesortchambertopreventaccidentallydroppingit.Workinginsidethesortchamber,wipebothplatesinonedirectionwithsterilewateroralcoholswipetoptobottom.Cleanthesidesandfloorofthesortchamberwherethewastegoes.Thesidewindowsneedtobecleanedforsidestreamevaluationdemonstratecleaningthesewindows.

5.INREADYMODE:Putthemachinebackinto“Ready”mode.Inthesame“PressureOptions”dialogbox,clickon“Ready”button.Themachinewillnowstartre-pressurizing.

6.Runthebleachcleaning(Cytometertab>“BleachClean”buttonontheribbon).Followtheprompts.Cleanwith15mLconical.

7.RuntheDIWaterclean(Cytometertab>“DIRinse”buttonontheribbon).Followtheprompts.Cleanwith15mLtube.You’renowgood-to-go.

8.(OPTIONAL)Whilecleaningisrunning,youcanstartmakinggraphsandapproximategatesforyourexperiments.Createdelements,plotsandapproximategatesbeforecollectingdatawillallowthemtobeavailableinallthetubes.Ifyouforgetthisstepyoucancopytheworksheetelementstothenexttube.

9.TheAerosolManagementSystemforthisunitisONLYonwhenyouareremovingasampleaftersortingortocleantheunitafteraclog.Turnontheunitusingthepowerbuttonbottomleftofunit.Hitthefootpedalonthefloortoactivatesuction.Leavetheunitevacuatingfor30-60seconds.THEUNITISOFFWHENYOUARESETTINGUPANDSORTING.ItisonlyactivewhenthedooristobeopenedafterBSL-2sortingorineventofaclog,thenturnitoffimmediately

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PleasepickonlyONEdyepercolumnumn.

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