演示文稿多色流式細(xì)胞調(diào)補(bǔ)償與圈門第一頁(yè)，共20頁(yè)。23-13536-00Rev.01（優(yōu)選）多色流式細(xì)胞調(diào)補(bǔ)償與圈門第二頁(yè)，共20頁(yè)。CompensationGoalRemovespilloversignalssothatsubpopulationMFIsagree第三頁(yè)，共20頁(yè)。DoesCompensationIncreaseDataSpread?UncompensatedCompensatedCompensationcorrectstheMFI,butcannotremoveallofthevariationincreaseintroducedbyspillover.However,undernormalconditionsthespreadinafluorescencemeasurementwithspilloverwilldecreasewhencompensationisapplied.Whenviewingdataonlogandbiexponentialplots,dataspreadappearstoincreasewhencompensationisapplied—thisisoftenavisualartifactduetothenon-linearscalingoftheplot.Spread第四頁(yè)，共20頁(yè)。CytometerSettingsWorkflowObtainCS&TsettingsApplyapplicationsettingsVerifysettingswithsampleCalculatecompensation第五頁(yè)，共20頁(yè)。CompensationforTandemDyesCompensationfortandemdyeconjugatescanvary,evenbetweentwoexperimentswiththesameantibody.Tandemdyesrequirecompensationthatis:lot-specificexperiment-specificlabel-specific第六頁(yè)，共20頁(yè)。CompensationRules—Part1Thefluorescenceemissionspectrum

ofcompensationcontrolsmustmatch

theexperimentreagents—thisis

especiallycriticalwithtandemreagents.Compensationcontrolnegativeandpositive

populationsmustbefromthesamecellorparticletype. Example:don’tuseaCD3–monocytenegativepopulationwithaCD3+lymphocytepositivepopulation.第七頁(yè)，共20頁(yè)。CompensationRules—Part2Compensationcontrolsmustbebrightenoughtoobtaingoodseparationbetweenthepositiveandnegativepopulations.Compensationcontrolsmustplacethepositivepopulationinthelinearrange.Whenusingcellsforcompensation

controls,increasethenumberof

eventstoatleast10,000pertube.WheneverMFItargetvalueschange,

reruncompensation.第八頁(yè)，共20頁(yè)。BDCompBeadsUsethesameantibodiesasintheexperimentalsamples.Createbrightanduniformpositivefluorescencepeaks.Avoidusinglimitedsample.Beadsarecoatedwithanti-mousekappa*.*BDCompBeadsarealsoavailablecoatedwithanti-ratkappaandanti-hamsterkappa.CompBeadAconvenientway

tocreateaccurate

single-colorcompensationcontrols第九頁(yè)，共20頁(yè)。UnstainedCompensationControlTubeWhenshouldIuseanunstainedcompensationcontroltube?InBDFACSDivasoftware:

Ifusingaseparateunstainedcontrol,selectthe

Includeseparateunstainedcontroltube/well

checkbox.Ifnotusingaseparateunstainedcontrol,clearthecheckboxandforeachparameterincludeaP3gateforthenegativepopulation.第十頁(yè)，共20頁(yè)。CompensationQCBiexponentialdisplayrevealscompensationproblems.OverCorrectBiexponential第十一頁(yè)，共20頁(yè)。CompensationDiscussionPointsHowoften?HowtoQCandadjustsettings?Postacquisition?Shouldcompensationcontrolsbetreatedthesameasexperimentalsamples?

(example:fixedandpermeabilized)第十二頁(yè)，共20頁(yè)。Controls第十三頁(yè)，共20頁(yè)。ChooseAppropriateControlsWhatWhyCytometersetupcontrols

BDCompBeadsEnsureconsistentsetupandcompensationGatingcontrols

FMO

Isotype

CombinedObtainreliablegatesforproblemmarkersBiologicalcontrols

Unstimulatedsamples

HealthydonorsMakeappropriatebiologicalcomparisonsandconclusions第十四頁(yè)，共20頁(yè)。GatingControlsFluorescence-Minus-One(FMO)controlIncludesalltestantibodiesexcepttheoneofinterest.Doesn’ttakebackgroundstainingintoaccount.Usefulinsettinggatesandconfirmingspilloverproblems.IsotypecontrolNon-specificantibodyofsameisotypeasthetestantibody.Doesn’ttakespilloverintoaccount.CombinedcontrolAlltestantibodiesexcepttheoneofinterest,whichisreplacedbyanisotypecontrol.Mightnotaccuratelyrepresentthebackgroundstainingofthetestantibody.第十五頁(yè)，共20頁(yè)。FMOExampleGatedonlymphs,CD3+CD4-Gatedonlymphs,CD3+CD4+Full9-colorcocktailFMOAmCyan第十六頁(yè)，共20頁(yè)。ComparisonofGatingControls第十七頁(yè)，共20頁(yè)。DataCollection第十八頁(yè)，共20頁(yè)。125,000lymphocytescol