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Rev 20200320 1 Sony MA900 Cell Sorter 70 and 100 µm chips are currently in use 130 chips are being updated release planned 2020 On First user of the Day Start Up Procedure: If the Instrument is on and calibrated skip to page 7 1. 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. First clean the sample and sort chambers with Cavicide. Do not spray inside the chamber as it may dirty the window optics that monitor the side streams. Soak a kim wipe and then clean all the surfaces inside the chamber except the sort plates. The sort plates can only be cleaned with clean water or 70% ethanol. To clean the plates carefully remove one plate by undoing the thumb screws. Leave the sort plate inside the sort chamber to prevent accidentally dropping it. Working inside the sort chamber, wipe both plates in one direction with alcohol swipe from top to bottom. Clean the base of the sort chamber, point out floor drain area to clean. 3. Fill sheath tank to below the upper weld line. Check the pressure release ring on top of the tank to be sure it is down in the groove on the top of relief valve. 4. Empty the waste tank, add bleach to maintain 10% for biosafety decontamination. Hold the metal fittings in the cap stable and turn the outer cap only to remove. Make sure the filter on the waste cap points up and is not allowed to get wet. There is a beaker on the floor to place it in to keep it oriented in the correct position. 5. Staff generally fills the the water, 70% ethanol and 10% bleach tank levels. Users need the sheath full and waste empty to run. 6. Power up the MA900. FACS Core Lab
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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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