多色流式細胞調(diào)補償與圈門

演示文稿多色流式細胞調(diào)補償與圈門目前一頁\總數(shù)二十頁\編于二十一點23-13536-00Rev.01（優(yōu)選）多色流式細胞調(diào)補償與圈門目前二頁\總數(shù)二十頁\編于二十一點CompensationGoalRemovespilloversignalssothatsubpopulationMFIsagree目前三頁\總數(shù)二十頁\編于二十一點DoesCompensationIncreaseDataSpread?UncompensatedCompensatedCompensationcorrectstheMFI,butcannotremoveallofthevariationincreaseintroducedbyspillover.However,undernormalconditionsthespreadinafluorescencemeasurementwithspilloverwilldecreasewhencompensationisapplied.Whenviewingdataonlogandbiexponentialplots,dataspreadappearstoincreasewhencompensationisapplied—thisisoftenavisualartifactduetothenon-linearscalingoftheplot.Spread目前四頁\總數(shù)二十頁\編于二十一點CytometerSettingsWorkflowObtainCS&TsettingsApplyapplicationsettingsVerifysettingswithsampleCalculatecompensation目前五頁\總數(shù)二十頁\編于二十一點CompensationforTandemDyesCompensationfortandemdyeconjugatescanvary,evenbetweentwoexperimentswiththesameantibody.Tandemdyesrequirecompensationthatis:lot-specificexperiment-specificlabel-specific目前六頁\總數(shù)二十頁\編于二十一點CompensationRules—Part1Thefluorescenceemissionspectrum

ofcompensationcontrolsmustmatch

theexperimentreagents—thisis

especiallycriticalwithtandemreagents.Compensationcontrolnegativeandpositive

populationsmustbefromthesamecellorparticletype. Example:don’tuseaCD3–monocytenegativepopulationwithaCD3+lymphocytepositivepopulation.目前七頁\總數(shù)二十頁\編于二十一點CompensationRules—Part2Compensationcontrolsmustbebrightenoughtoobtaingoodseparationbetweenthepositiveandnegativepopulations.Compensationcontrolsmustplacethepositivepopulationinthelinearrange.Whenusingcellsforcompensation

controls,increasethenumberof

eventstoatleast10,000pertube.WheneverMFItargetvalueschange,

reruncompensation.目前八頁\總數(shù)二十頁\編于二十一點BDCompBeadsUsethesameantibodiesasintheexperimentalsamples.Createbrightanduniformpositivefluorescencepeaks.Avoidusinglimitedsample.Beadsarecoatedwithanti-mousekappa*.*BDCompBeadsarealsoavailablecoatedwithanti-ratkappaandanti-hamsterkappa.CompBeadAconvenientway

tocreateaccurate

single-colorcompensationcontrols目前九頁\總數(shù)二十頁\編于二十一點UnstainedCompensationControlTubeWhenshouldIuseanunstainedcompensationcontroltube?InBDFACSDivasoftware:

Ifusingaseparateunstainedcontrol,selectthe

Includeseparateunstainedcontroltube/well

checkbox.Ifnotusingaseparateunstainedcontrol,clearthecheckboxandforeachparameterincludeaP3gateforthenegativepopulation.目前十頁\總數(shù)二十頁\編于二十一點CompensationQCBiexponentialdisplayrevealscompensationproblems.OverCorrectBiexponential目前十一頁\總數(shù)二十頁\編于二十一點CompensationDiscussionPointsHowoften?HowtoQCandadjustsettings?Postacquisition?Shouldcompensationcontrolsbetreatedthesameasexperimentalsamples?

(example:fixedandpermeabilized)目前十二頁\總數(shù)二十頁\編于二十一點Controls目前十三頁\總數(shù)二十頁\編于二十一點ChooseAppropriateControlsWhatWhyCytometersetupcontrols

BDCompBeadsEnsureconsistentsetupandcompensationGatingcontrols

FMO

Isotype

CombinedObtainreliablegatesforproblemmarkersBiologicalcontrols

Unstimulatedsamples

HealthydonorsMakeappropriatebiologicalcomparisonsandconclusions目前十四頁\總數(shù)二十頁\編于二十一點GatingControlsFluorescence-Minus-One(FMO)controlIncludesalltestantibodiesexcepttheoneofinterest.Doesn’ttakebackgroundstainingintoaccount.Usefulinsettinggatesandconfirmingspilloverproblems.IsotypecontrolNon-specificantibodyofsameisotypeasthetestantibody.Doesn’ttakespilloverintoaccount.CombinedcontrolAlltestantibodiesexcepttheoneofinterest,whichisreplacedbyanisotypecontrol.Mightnotaccuratelyrepresentthebackgroundstainingofthetestantibody.目前十五頁\總數(shù)二十頁\編于二十一點FMOExampleGatedonlymphs,CD3+CD4-Gatedonlymphs,CD3+CD4+Full9-colorcocktailFMOAmCyan目前十六頁\總數(shù)二十頁\編于二十一點ComparisonofGatingControls目前十七頁\總數(shù)二十頁\編于二十一點DataCollection目前十八頁\總數(shù)二十頁\編于二十一點125,000lymphocytescollected20,000lymphocytes

collectedNumberofEventsvsMeasurementPrecisionCD4+TcellsCD8+Tcells14events=0.14%73events=0.34%8events=0.23%51events=0.09%目前十九頁\總數(shù)二十頁\編于二十一點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