Compensation,Controls,

andDataCollection23-13536-00Rev.01多色流式細(xì)胞FACS調(diào)補(bǔ)償與圈門專家講座第1頁CompensationGoalRemovespilloversignalssothatsubpopulationMFIsagree223-13536-00Rev.01多色流式細(xì)胞FACS調(diào)補(bǔ)償與圈門專家講座第2頁DoesCompensationIncreaseDataSpread?UncompensatedCompensatedCompensationcorrectstheMFI,butcannotremoveallofthevariationincreaseintroducedbyspillover.However,undernormalconditionsthespreadinafluorescencemeasurementwithspilloverwilldecreasewhencompensationisapplied.Whenviewingdataonlogandbiexponentialplots,dataspreadappearstoincreasewhencompensationisapplied—thisisoftenavisualartifactduetothenon-linearscalingoftheplot.Spread323-13536-00Rev.01多色流式細(xì)胞FACS調(diào)補(bǔ)償與圈門專家講座第3頁CytometerSettingsWorkflowObtainCS&TsettingsApplyapplicationsettingsVerifysettingswithsampleCalculatecompensation423-13536-00Rev.01多色流式細(xì)胞FACS調(diào)補(bǔ)償與圈門專家講座第4頁CompensationforTandemDyesCompensationfortandemdyeconjugatescanvary,evenbetweentwoexperimentswiththesameantibody.Tandemdyesrequirecompensationthatis:lot-specificexperiment-specificlabel-specific523-13536-00Rev.01多色流式細(xì)胞FACS調(diào)補(bǔ)償與圈門專家講座第5頁CompensationRules—Part1Thefluorescenceemissionspectrum

ofcompensationcontrolsmustmatch

theexperimentreagents—thisis

especiallycriticalwithtandemreagents.Compensationcontrolnegativeandpositive

populationsmustbefromthesamecellorparticletype. Example:don’tuseaCD3–monocytenegativepopulationwithaCD3+lymphocytepositivepopulation.623-13536-00Rev.01多色流式細(xì)胞FACS調(diào)補(bǔ)償與圈門專家講座第6頁CompensationRules—Part2Compensationcontrolsmustbebrightenoughtoobtaingoodseparationbetweenthepositiveandnegativepopulations.Compensationcontrolsmustplacethepositivepopulationinthelinearrange.Whenusingcellsforcompensation

controls,increasethenumberof

eventstoatleast10,000pertube.WheneverMFItargetvalueschange,

reruncompensation.723-13536-00Rev.01多色流式細(xì)胞FACS調(diào)補(bǔ)償與圈門專家講座第7頁BDCompBeadsUsethesameantibodiesasintheexperimentalsamples.Createbrightanduniformpositivefluorescencepeaks.Avoidusinglimitedsample.Beadsarecoatedwithanti-mousekappa*.*BDCompBeadsarealsoavailablecoatedwithanti-ratkappaandanti-hamsterkappa.CompBeadAconvenientway

tocreateaccurate

single-colorcompensationcontrols823-13536-00Rev.01多色流式細(xì)胞FACS調(diào)補(bǔ)償與圈門專家講座第8頁UnstainedCompensationControlTubeWhenshouldIuseanunstainedcompensationcontroltube?InBDFACSDivasoftware:

Ifusingaseparateunstainedcontrol,selectthe

Includeseparateunstainedcontroltube/well

checkbox.Ifnotusingaseparateunstainedcontrol,clearthecheckboxandforeachparameterincludeaP3gateforthenegativepopulation.923-13536-00Rev.01多色流式細(xì)胞FACS調(diào)補(bǔ)償與圈門專家講座第9頁CompensationQCBiexponentialdisplayrevealscompensationproblems.OverCorrectBiexponential1023-13536-00Rev.01多色流式細(xì)胞FACS調(diào)補(bǔ)償與圈門專家講座第10頁CompensationDiscussionPointsHowoften?HowtoQCandadjustsettings?Postacquisition?Shouldcompensationcontrolsbetreatedthesameasexperimentalsamples?

(example:fixedandpermeabilized)1123-13536-00Rev.01多色流式細(xì)胞FACS調(diào)補(bǔ)償與圈門專家講座第11頁Controls1223-13536-00Rev.01多色流式細(xì)胞FACS調(diào)補(bǔ)償與圈門專家講座第12頁ChooseAppropriateControlsWhatWhyCytometersetupcontrols

BDCompBeadsEnsureconsistentsetupandcompensationGatingcontrols

FMO

Isotype

CombinedObtainreliablegatesforproblemmarkersBiologicalcontrols

Unstimulatedsamples

HealthydonorsMakeappropriatebiologicalcomparisonsandconclusions1323-13536-00Rev.01多色流式細(xì)胞FACS調(diào)補(bǔ)償與圈門專家講座第13頁GatingControlsFluorescence-Minus-One(FMO)controlIncludesalltestantibodiesexcepttheoneofinterest.Doesn’ttakebackgroundstainingintoaccount.Usefulinsettinggatesandconfirmingspilloverproblems.IsotypecontrolNon-specificantibodyofsameisotypeasthetestantibody.Doesn’ttakespilloverintoaccount.CombinedcontrolAlltestantibodiesexcepttheoneofinterest,whichisreplacedbyanisotypecontrol.Mightnotaccuratelyrepresentthebackgroundstainingofthetestantibody.1423-13536-00Rev.01多色流式細(xì)胞FACS調(diào)補(bǔ)償與圈門專家講座第14頁FMOExampleGatedonlymphs,CD3+CD4-Gatedonlymphs,CD3+CD4+Full9-colorcocktailFMOAmCyan1523-13536-00Rev.01多色流式細(xì)胞FACS調(diào)補(bǔ)償與圈門專家講座第15頁ComparisonofGatingControls1623-13536-00Rev.01多色流式細(xì)胞FACS調(diào)補(bǔ)償與圈門專家講座第16頁DataCollection1723-13536-00Rev.01多色流式細(xì)胞FACS調(diào)補(bǔ)償與圈門專家講座第17頁125,000lymphocytescollected20,000lymphocytes

collectedNumberofEventsvsMeasurementPrecisionCD4+TcellsCD8+Tcells14events=0.14%73events=0.34%8events=0.23%51events=0.09%1823-13536-00Rev.01多色流式細(xì)胞FACS調(diào)補(bǔ)償與圈門專家講座第18頁StatisticalSignificanceofResultsDeterminingtheNumberofEventstoCollect

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

160,00