多色流式细胞(FACS)调补偿与圈门课件

Compensation,Controls,

andDataCollection多色流式细胞(FACS)调补偿与圈门CompensationGoalRemovespilloversignalssothatsubpopulationMFIsagree2多色流式细胞(FACS)调补偿与圈门DoesCompensationIncreaseDataSpread?UncompensatedCompensatedCompensationcorrectstheMFI,butcannotremoveallofthevariationincreaseintroducedbyspillover.However,undernormalconditionsthespreadinafluorescencemeasurementwithspilloverwilldecreasewhencompensationisapplied.Whenviewingdataonlogandbiexponentialplots,dataspreadappearstoincreasewhencompensationisapplied—thisisoftenavisualartifactduetothenon-linearscalingoftheplot.Spread3多色流式细胞(FACS)调补偿与圈门CytometerSettingsWorkflowObtainCS&TsettingsApplyapplicationsettingsVerifysettingswithsampleCalculatecompensation4多色流式细胞(FACS)调补偿与圈门CompensationforTandemDyesCompensationfortandemdyeconjugatescanvary,evenbetweentwoexperimentswiththesameantibody.Tandemdyesrequirecompensationthatis:lot-specificexperiment-specificlabel-specific5多色流式细胞(FACS)调补偿与圈门CompensationRules—Part1Thefluorescenceemissionspectrum

ofcompensationcontrolsmustmatch

theexperimentreagents—thisis

especiallycriticalwithtandemreagents.Compensationcontrolnegativeandpositive

populationsmustbefromthesamecellorparticletype. Example:don’tuseaCD3–monocytenegativepopulationwithaCD3+lymphocytepositivepopulation.6多色流式细胞(FACS)调补偿与圈门CompensationRules—Part2Compensationcontrolsmustbebrightenoughtoobtaingoodseparationbetweenthepositiveandnegativepopulations.Compensationcontrolsmustplacethepositivepopulationinthelinearrange.Whenusingcellsforcompensation

controls,increasethenumberof

eventstoatleast10,000pertube.WheneverMFItargetvalueschange,

reruncompensation.7多色流式细胞(FACS)调补偿与圈门BDCompBeadsUsethesameantibodiesasintheexperimentalsamples.Createbrightanduniformpositivefluorescencepeaks.Avoidusinglimitedsample.Beadsarecoatedwithanti-mousekappa*.*BDCompBeadsarealsoavailablecoatedwithanti-ratkappaandanti-hamsterkappa.CompBeadAconvenientway

tocreateaccurate

single-colorcompensationcontrols8多色流式细胞(FACS)调补偿与圈门UnstainedCompensationControlTubeWhenshouldIuseanunstainedcompensationcontroltube?InBDFACSDivasoftware:

Ifusingaseparateunstainedcontrol,selectthe

Includeseparateunstainedcontroltube/well

checkbox.Ifnotusingaseparateunstainedcontrol,clearthecheckboxandforeachparameterincludeaP3gateforthenegativepopulation.9多色流式细胞(FACS)调补偿与圈门CompensationQCBiexponentialdisplayrevealscompensationproblems.OverCorrectBiexponential10多色流式细胞(FACS)调补偿与圈门CompensationDiscussionPointsHowoften?HowtoQCandadjustsettings?Postacquisition?Shouldcompensationcontrolsbetreatedthesameasexperimentalsamples?

(example:fixedandpermeabilized)11多色流式细胞(FACS)调补偿与圈门Controls12多色流式细胞(FACS)调补偿与圈门ChooseAppropriateControlsWhatWhyCytometersetupcontrols

BDCompBeadsEnsureconsistentsetupandcompensationGatingcontrols

FMO

Isotype

CombinedObtainreliablegatesforproblemmarkersBiologicalcontrols

Unstimulatedsamples

HealthydonorsMakeappropriatebiologicalcomparisonsandconclusions13多色流式细胞(FACS)调补偿与圈门GatingControlsFluorescence-Minus-One(FMO)controlIncludesalltestantibodiesexcepttheoneofinterest.Doesn’ttakebackgroundstainingintoaccount.Usefulinsettinggatesandconfirmingspilloverproblems.IsotypecontrolNon-specificantibodyofsameisotypeasthetestantibody.Doesn’ttakespilloverintoaccount.CombinedcontrolAlltestantibodiesexcepttheoneofinterest,whichisreplacedbyanisotypecontrol.Mightnotaccuratelyrepresentthebackgroundstainingofthetestantibody.14多色流式细胞(FACS)调补偿与圈门FMOExampleGatedonlymphs,CD3+CD4-Gatedonlymphs,CD3+CD4+Full9-colorcocktailFMOAmCyan15多色流式细胞(FACS)调补偿与圈门ComparisonofGatingControls16多色流式细胞(FACS)调补偿与圈门DataCollection17多色流式细胞(FACS)调补偿与圈门125,000lymphocytescollected20,000lymphocytes

collectedNumberofEventsvsMeasurementPrecisionCD4+TcellsCD8+Tcells14events=0.14%73events=0.34%8events=0.23%51events=0.09%18多色流式细胞(FACS)调补偿与圈门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

160,000450,0000.08

0.2

17,00046,0000.1

0.2

26,00072,000

0.10.119多色流式细胞(FACS)调补偿与圈门DataCollectionDiscussionStorageGatesandStoppingGatesGlobalWorksheetsvsNormalWorksheetsTemplates20多色流式细胞(FACS)调补偿与圈门ExperimentSetupand

RecordSam