用流式细胞仪分析脂肪细胞课件

AnalysisandIsolationofAdipocytesbyFlowCytometry,YangLI2016-04-05,MajkaSM,MillerHL,HelmKM,etal.AnalysisandIsolationofAdipocytesbyFlowCytometry.Methodsinenzymology.2014;537:281-296.doi:10.1016/B978-0-12-411619-1.00015-X.,1,学习交流PPT,2,CONTENTS,BackgroundAimoftheresearchDifficultySolution,2,学习交流PPT,Background,Whatareadipocytes(fatcells)?Majorcellsintheadiposetissue.,3,3,学习交流PPT,Background,Lipiddroplet,Twokindsofadipocytesinhuman,4,4,学习交流PPT,Prospectsofadipocytebiology,Background,Lipidstore,InhibiteatingIncreasemetabolism,Leptin,5,5,学习交流PPT,Prospectsofadipocytebiology,Background,Adipokines,Involvedinenergymetabolism,Lipidstore,6,6,学习交流PPT,ProspectsofadipocytebiologySpecificallyfromadiposetissue,Background,Lipidstore,Adipokines,Energymetabolism,7,7,学习交流PPT,ProspectsofadipocytebiologyAdipokinesplayansignificantroleinbiologicalfunction.,Background,8,8,学习交流PPT,Background,FlowCytometry(FCM),Principlediagram,9,9,学习交流PPT,Background,Volume,Morphologicalcomplexityofcells,TotalDNA/RNAcontent,Apoptosis,Cellviability,.,10,10,学习交流PPT,Aimoftheresearch,Amethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.,Analysis,Flowcytometry(FCM)Confocallaserscanningmicroscope(CLSM),Isolation,Fluorescence-activatedcellsorting(FACS),11,11,学习交流PPT,Aimoftheresearch,Analysis,Flowcytometry(FCM)Confocallaserscanningmicroscope(CLSM),BeckmanCoulter-MoFloXDP,Analysisrate:100,000events/sec.Sortrate:70,000events/sec.,12,Amethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.,12,学习交流PPT,Aimoftheresearch,Analysis,Flowcytometry(FCM)Confocallaserscanningmicroscope(CLSM),Quantitive,Highresolutionimaging,13,Amethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.,13,学习交流PPT,Aimoftheresearch,Analysis,Flowcytometry(FCM)Confocallaserscanningmicroscope(CLSM),ImagingFlowCytometer(Amnis),12Imagesforeverycell,Quantitive,Highresolutionimaging,14,Amethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.,14,学习交流PPT,Aimoftheresearch,Isolation,Fluorescence-activatedcellsorting(FACS),Apassingcellcanbeselectivelyadddifferentcharge,fallingintodifferentcontainers.,15,Amethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.,15,学习交流PPT,Difficulty,Adipocytesarelargeandfragile,50200mEasytobebroken,whentheflowrate(pressure)islarge.,16,16,学习交流PPT,Difficulty,Adipocytesarelargeandfragile,50200mEasytobebroken,whentheflowrate(pressure)islarge.,Contaminants,Stromal/vascularcellsAggregatesofadipocytesFreelipiddropletsOtherdebris,17,17,学习交流PPT,Solution,Adipocytesarelargeandfragile,50200musingflowcytometrywithlargerinternaldiameterofthefluidics(150to250m).EasytobebrokencontrolFlowpressuresto510psi,slowbutnotbroken.Goodforisolation.,18,18,学习交流PPT,Solution,Contaminants,Stromal/vascularcellsAggregatesofadipocytesFreelipiddropletsOtherdebrisExclusionofContaminants(4steps),19,19,学习交流PPT,Solution,CollagenaseDigestionFiltrationUndigestedtissuefragmentsareexcluded.CentrifugationSeparatedfromstromal/vascularcellsFragileUserockingratherthanvortexing,Preparationofsinglecellsuspensionoffree-floatingadipocytes,20,20,学习交流PPT,Solution,Step1.Initialseparationbysize,Adipocytes(50200m)biggerthanstromal/vascularcells(20m)higherinForwardScatter(FSC)ThepresenceofhighlyrefractilelipiddropletsinadipocytehigherinSSCOverlapneedfurtherseparationSmalladipocytesignored(entireanalyzeifnecessary),21,21,学习交流PPT,Solution,Contaminants,Stromal/vascularcellsAggregatesofadipocytesFreelipiddropletsOtherdebrisExclusionofContaminants(4steps),22,22,学习交流PPT,Solution,Step2.Exclusionofaggregatesandselectionofsinglets,Aggregatescanbeidentifiedbydotplot(WidthorArea).3parameters:Heightvoltage,Widthpassingtime(size),Area=HxWWhenaggregates(doublets)pass,spendmoretime,Wincreased,23,23,学习交流PPT,Solution,Step2.Exclusionofaggregatesandselectionofsinglets,StainnucleibyDyeCycleViolet(DCV)fluorescence.Singlecellscanbeeasilygatedfromaggregates&otherdebrisThisstepcouldntexcluderemainingstromal/vascularcells.,24,24,学习交流PPT,Solution,Step3.Exclusionofsinglestromal/vascularcells,StainthelipiddropletsbyLipidTOXDeepRed(LpdTX)PositiveSelectionofeventscontaininglipiddroplets,25,25,学习交流PPT,Solution,Step4:Exclusionofremainingstromalcontaminants,Asmallprobabilityevent:Somestromalcellsmayadheretothefreelipiddroplets.Theremainingstromalcontaminantsbearingthefluorescentantibodies(phycoerythrin(PE)-conjugated)tostromallineagemarkersarestainedtoexclude.(Negativeselection),Negativeselection,26