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多色流式细胞(FACS)调补偿与圈门.ppt

多色流式细胞(FACS)调补偿与圈门.ppt

ID：49748840

大小：2.72 MB

页数：22页

时间：2020-03-01

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1、Compensation,Controls, andDataCollection23-13536-00Rev.01CompensationGoalRemovespilloversignalssothatsubpopulationMFIsagree223-13536-00Rev.01DoesCompensationIncreaseDataSpread?UncompensatedCompensatedCompensationcorrectstheMFI,butcannotremoveallofthevariationincreasein

2、troducedbyspillover.However,undernormalconditionsthespreadinafluorescencemeasurementwithspilloverwilldecreasewhencompensationisapplied.Whenviewingdataonlogandbiexponentialplots,dataspreadappearstoincreasewhencompensationisapplied—thisisoftenavisualartifactduetothenon-l

3、inearscalingoftheplot.Spread323-13536-00Rev.01CytometerSettingsWorkflowObtainCS&TsettingsApplyapplicationsettingsVerifysettingswithsampleCalculatecompensation423-13536-00Rev.01CompensationforTandemDyesCompensationfortandemdyeconjugatescanvary,evenbetweentwoexperimentsw

4、iththesameantibody.Tandemdyesrequirecompensationthatis:lot-specificexperiment-specificlabel-specific523-13536-00Rev.01CompensationRules—Part1Thefluorescenceemissionspectrum ofcompensationcontrolsmustmatch theexperimentreagents—thisis especiallycriticalwithtandemreagent

5、s.Compensationcontrolnegativeandpositive populationsmustbefromthesamecellorparticletype.Example:don’tuseaCD3–monocytenegativepopulationwithaCD3+lymphocytepositivepopulation.623-13536-00Rev.01CompensationRules—Part2Compensationcontrolsmustbebrightenoughtoobtaingoodsepar

6、ationbetweenthepositiveandnegativepopulations.Compensationcontrolsmustplacethepositivepopulationinthelinearrange.Whenusingcellsforcompensation controls,increasethenumberof eventstoatleast10,000pertube.WheneverMFItargetvalueschange, reruncompensation.723-13536-00Rev.01B

7、DCompBeadsUsethesameantibodiesasintheexperimentalsamples.Createbrightanduniformpositivefluorescencepeaks.Avoidusinglimitedsample.Beadsarecoatedwithanti-mousekappa*.*BDCompBeadsarealsoavailablecoatedwithanti-ratkappaandanti-hamsterkappa.CompBeadAconvenientway tocreateac

8、curatesingle-colorcompensationcontrols823-13536-00Rev.01UnstainedCompensationControlTubeWhenshouldIuseanunstainedcomp

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