多色流式细胞调补偿与圈门

演示文稿多色流式细胞调补偿与圈门目前一页\总数二十页\编于二十一点23-13536-00Rev.01（优选）多色流式细胞调补偿与圈门目前二页\总数二十页\编于二十一点CompensationGoalRemovespilloversignalssothatsubpopulationMFIsagree目前三页\总数二十页\编于二十一点DoesCompensationIncreaseDataSpread?UncompensatedCompensatedCompensationcorrectstheMFI,butcannotremoveallofthevariationincreaseintroducedbyspillover.However,undernormalconditionsthespreadinafluorescencemeasurementwithspilloverwilldecreasewhencompensationisapplied.Whenviewingdataonlogandbiexponentialplots,dataspreadappearstoincreasewhencompensationisapplied—thisisoftenavisualartifactduetothenon-linearscalingoftheplot.Spread目前四页\总数二十页\编于二十一点CytometerSettingsWorkflowObtainCS&TsettingsApplyapplicationsettingsVerifysettingswithsampleCalculatecompensation目前五页\总数二十页\编于二十一点CompensationforTandemDyesCompensationfortandemdyeconjugatescanvary,evenbetweentwoexperimentswiththesameantibody.Tandemdyesrequirecompensationthatis:lot-specificexperiment-specificlabel-specific目前六页\总数二十页\编于二十一点CompensationRules—Part1Thefluorescenceemissionspectrum

ofcompensationcontrolsmustmatch

theexperimentreagents—thisis

especiallycriticalwithtandemreagents.Compensationcontrolnegativeandpositive

populationsmustbefromthesamecellorparticletype. Example:don’tuseaCD3–monocytenegativepopulationwithaCD3+lymphocytepositivepopulation.目前七页\总数二十页\编于二十一点CompensationRules—Part2Compensationcontrolsmustbebrightenoughtoobtaingoodseparationbetweenthepositiveandnegativepopulations.Compensationcontrolsmustplacethepositivepopulationinthelinearrange.Whenusingcellsforcompensation

controls,increasethenumberof

eventstoatleast10,000pertube.WheneverMFItargetvalueschange,

reruncompensation.目前八页\总数二十页\编于二十一点BDCompBeadsUsethesameantibodiesasintheexperimentalsamples.Createbrightanduniformpositivefluorescencepeaks.Avoidusinglimitedsample.Beadsarecoatedwithanti-mousekappa*.*BDCompBeadsarealsoavailablecoatedwithanti-ratkappaandanti-hamsterkappa.CompBeadAconvenientway

tocreateaccurate

single-colorcompensationcontrols目前九页\总数二十页\编于二十一点UnstainedCompensationControlTubeWhenshouldIuseanunstainedcompensationcontroltube?InBDFACSDivasoftware:

Ifusingaseparateunstainedcontrol,selectthe

Includeseparateunstainedcontroltube/well

checkbox.Ifnotusingaseparateunstainedcontrol,clearthecheckboxandforeachparameterincludeaP3gateforthenegativepopulation.目前十页\总数二十页\编于二十一点CompensationQCBiexponentialdisplayrevealscompensationproblems.OverCorrectBiexponential目前十一页\总数二十页\编于二十一点CompensationDiscussionPointsHowoften?HowtoQCandadjustsettings?Postacquisition?Shouldcompensationcontrolsbetreatedthesameasexperimentalsamples?

(example:fixedandpermeabilized)目前十二页\总数二十页\编于二十一点Controls目前十三页\总数二十页\编于二十一点ChooseAppropriateControlsWhatWhyCytometersetupcontrols

BDCompBeadsEnsureconsistentsetupandcompensationGatingcontrols

FMO

Isotype

CombinedObtainreliablegatesforproblemmarkersBiologicalcontrols

Unstimulatedsamples

HealthydonorsMakeappropriatebiologicalcomparisonsandconclusions目前十四页\总数二十页\编于二十一点GatingControlsFluorescence-Minus-One(FMO)controlIncludesalltestantibodiesexcepttheoneofinterest.Doesn’ttakebackgroundstainingintoaccount.Usefulinsettinggatesandconfirmingspilloverproblems.IsotypecontrolNon-specificantibodyofsameisotypeasthetestantibody.Doesn’ttakespilloverintoaccount.CombinedcontrolAlltestantibodiesexcepttheoneofinterest,whichisreplacedbyanisotypecontrol.Mightnotaccuratelyrepresentthebackgroundstainingofthetestantibody.目前十五页\总数二十页\编于二十一点FMOExampleGatedonlymphs,CD3+CD4-Gatedonlymphs,CD3+CD4+Full9-colorcocktailFMOAmCyan目前十六页\总数二十页\编于二十一点ComparisonofGatingControls目前十七页\总数二十页\编于二十一点DataCollection目前十八页\总数二十页\编于二十一点125,000lymphocytescollected20,000lymphocytes

collectedNumberofEventsvsMeasurementPrecisionCD4+TcellsCD8+Tcells14events=0.14%73events=0.34%8events=0.23%51events=0.09%目前十九页\总数二十页\编于二十一点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