多色流式細(xì)胞(FACS)調(diào)補償與圈門

Compensation,Controls,

andDataCollection整理版pptCompensationGoalRemovespilloversignalssothatsubpopulationMFIsagree2整理版pptDoesCompensationIncreaseDataSpread?UncompensatedCompensatedCompensationcorrectstheMFI,butcannotremoveallofthevariationincreaseintroducedbyspillover.However,undernormalconditionsthespreadinafluorescencemeasurementwithspilloverwilldecreasewhencompensationisapplied.Whenviewingdataonlogandbiexponentialplots,dataspreadappearstoincreasewhencompensationisapplied—thisisoftenavisualartifactduetothenon-linearscalingoftheplot.Spread3整理版pptCytometerSettingsWorkflowObtainCS&TsettingsApplyapplicationsettingsVerifysettingswithsampleCalculatecompensation4整理版pptCompensationforTandemDyesCompensationfortandemdyeconjugatescanvary,evenbetweentwoexperimentswiththesameantibody.Tandemdyesrequirecompensationthatis:lot-specificexperiment-specificlabel-specific5整理版pptCompensationRules—Part1Thefluorescenceemissionspectrum

ofcompensationcontrolsmustmatch

theexperimentreagents—thisis

especiallycriticalwithtandemreagents.Compensationcontrolnegativeandpositive

populationsmustbefromthesamecellorparticletype. Example:don’tuseaCD3–monocytenegativepopulationwithaCD3+lymphocytepositivepopulation.6整理版pptCompensationRules—Part2Compensationcontrolsmustbebrightenoughtoobtaingoodseparationbetweenthepositiveandnegativepopulations.Compensationcontrolsmustplacethepositivepopulationinthelinearrange.Whenusingcellsforcompensation

controls,increasethenumberof

eventstoatleast10,000pertube.WheneverMFItargetvalueschange,

reruncompensation.7整理版pptBDCompBeadsUsethesameantibodiesasintheexperimentalsamples.Createbrightanduniformpositivefluorescencepeaks.Avoidusinglimitedsample.Beadsarecoatedwithanti-mousekappa*.*BDCompBeadsarealsoavailablecoatedwithanti-ratkappaandanti-hamsterkappa.CompBeadAconvenientway

tocreateaccurate

single-colorcompensationcontrols8整理版pptUnstainedCompensationControlTubeWhenshouldIuseanunstainedcompensationcontroltube?InBDFACSDivasoftware:

Ifusingaseparateunstainedcontrol,selectthe

Includeseparateunstainedcontroltube/well

checkbox.Ifnotusingaseparateunstainedcontrol,clearthecheckboxandforeachparameterincludeaP3gateforthenegativepopulation.9整理版pptCompensationQCBiexponentialdisplayrevealscompensationproblems.OverCorrectBiexponential10整理版pptCompensationDiscussionPointsHowoften?HowtoQCandadjustsettings?Postacquisition?Shouldcompensationcontrolsbetreatedthesameasexperimentalsamples?

(example:fixedandpermeabilized)11整理版pptControls12整理版pptChooseAppropriateControlsWhatWhyCytometersetupcontrols

BDCompBeadsEnsureconsistentsetupandcompensationGatingcontrols

FMO

Isotype

CombinedObtainreliablegatesforproblemmarkersBiologicalcontrols

Unstimulatedsamples

HealthydonorsMakeappropriatebiologicalcomparisonsandconclusions13整理版pptGatingControlsFluorescence-Minus-One(FMO)controlIncludesalltestantibodiesexcepttheoneofinterest.Doesn’ttakebackgroundstainingintoaccount.Usefulinsettinggatesandconfirmingspilloverproblems.IsotypecontrolNon-specificantibodyofsameisotypeasthetestantibody.Doesn’ttakespilloverintoaccount.CombinedcontrolAlltestantibodiesexcepttheoneofinterest,whichisreplacedbyanisotypecontrol.Mightnotaccuratelyrepresentthebackgroundstainingofthetestantibody.14整理版pptFMOExampleGatedonlymphs,CD3+CD4-Gatedonlymphs,CD3+CD4+Full9-colorcocktailFMOAmCyan15整理版pptComparisonofGatingControls16整理版pptDataCollection17整理版ppt125,000lymphocytescollected20,000lymphocytes

collectedNumberofEventsvsMeasurementPrecisionCD4+TcellsCD8+Tcells14events=0.14%73events=0.34%8events=0.23%51events=0.09%18整理版pptStatisticalSignificanceofResultsDeterminingtheNumberofEventstoCollect

NumberofRelevantEventstoCollect

%Background(False+)

Lowest%Positive

90%power,p<0.05

99%power,p<0.005

0.01

0.02

260,000720,0000.01

0.05

32,00090,0000.01

12,00032,0000.02

0.05

67,000190,0000.02

16,00045,0000.03

0.05

170,000480,0000.03

0.1

23,00063,0000.04

0.1

33,00093,0000.05

0.1

52,000140,0000.06

0.1

86,000240,0000.07

0.1

160,000450,0000.08

0.2

17,00046,0000.1

0.2

26,00072,000

0.10.119整理版pptDataCollectionDiscussionStorageGatesandStoppingGatesGlobalWorksheetsvsNormalWorksheetsTemplates20整理版pptExperimen