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Rev20200320
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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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