用流式细胞仪分析脂肪细胞ppt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 2 CONTENTS BackgroundAimoftheresearchDifficultySolution Background Whatareadipocytes fatcells Majorcellsintheadiposetissue 3 Background Lipiddroplet Twokindsofadipocytesinhuman 4 Prospectsofadipocytebiology Background Lipidstore InhibiteatingIncreasemetabolism Leptin 5 Prospectsofadipocytebiology Background Adipokines Involvedinenergymetabolism Lipidstore 6 ProspectsofadipocytebiologySpecificallyfromadiposetissue Background Lipidstore Adipokines Energymetabolism 7 ProspectsofadipocytebiologyAdipokinesplayansignificantroleinbiologicalfunction Background 8 Background FlowCytometry FCM Principlediagram 9 Background Volume Morphologicalcomplexityofcells TotalDNA RNAcontent Apoptosis Cellviability 10 Aimoftheresearch Amethodforanalysisandisolationfree floatingadipocytesbyFlowCytometry Analysis Flowcytometry FCM Confocallaserscanningmicroscope CLSM Isolation Fluorescence activatedcellsorting FACS 11 Aimoftheresearch Analysis Flowcytometry FCM Confocallaserscanningmicroscope CLSM BeckmanCoulter MoFlo XDP Analysisrate 100 000events sec Sortrate 70 000events sec 12 Amethodforanalysisandisolationfree floatingadipocytesbyFlowCytometry Aimoftheresearch Analysis Flowcytometry FCM Confocallaserscanningmicroscope CLSM Quantitive Highresolutionimaging 13 Amethodforanalysisandisolationfree floatingadipocytesbyFlowCytometry Aimoftheresearch Analysis Flowcytometry FCM Confocallaserscanningmicroscope CLSM ImagingFlowCytometer Amnis 12Imagesforeverycell Quantitive Highresolutionimaging 14 Amethodforanalysisandisolationfree floatingadipocytesbyFlowCytometry Aimoftheresearch Isolation Fluorescence activatedcellsorting FACS Apassingcellcanbeselectivelyadddifferentcharge fallingintodifferentcontainers 15 Amethodforanalysisandisolationfree floatingadipocytesbyFlowCytometry Difficulty Adipocytesarelargeandfragile 50 200 mEasytobebroken whentheflowrate pressure islarge 16 Difficulty Adipocytesarelargeandfragile 50 200 mEasytobebroken whentheflowrate pressure islarge Contaminants Stromal vascularcellsAggregatesofadipocytesFreelipiddropletsOtherdebris 17 Solution Adipocytesarelargeandfragile 50 200 m usingflowcytometrywithlargerinternaldiameterofthefluidics 150to250 m Easytobebroken controlFlowpressuresto5 10psi slowbutnotbroken Goodforisolation 18 Solution Contaminants Stromal vascularcellsAggregatesofadipocytesFreelipiddropletsOtherdebris ExclusionofContaminants 4steps 19 Solution Collagenase DigestionFiltration Undigestedtissuefragmentsareexcluded Centrifugation Separatedfromstromal vascularcellsFragile Userockingratherthanvortexing Preparationofsinglecellsuspensionoffree floatingadipocytes 20 Solution Step1 Initialseparation bysize Adipocytes 50 200 m biggerthanstromal vascularcells 20 m higherinForwardScatter FSC Thepresenceofhighlyrefractilelipiddropletsinadipocyte higherinSSCOverlap needfurtherseparationSmalladipocytes ignored entireanalyzeifnecessary 21 Solution Contaminants Stromal vascularcellsAggregatesofadipocytesFreelipiddropletsOtherdebris ExclusionofContaminants 4steps 22 Solution Step2 Exclusionofaggregatesandselectionofsinglets Aggregatescanbeidentifiedbydotplot WidthorArea 3parameters Height voltage Width passingtime size Area HxWWhenaggregates doublets pass spendmoretime Wincreased 23 Solution Step2 Exclusionofaggregatesandselectionofsinglets StainnucleibyDyeCycleViolet DCV fluorescence Singlecellscanbeeasilygatedfromaggregates otherdebrisThisstepcouldn texcluderemainingstromal vascularcells 24 Solution Step3 Exclusionofsinglestromal vascularcells StainthelipiddropletsbyLipidTOXDeepRed LpdTX PositiveSelectionofeventscontaininglipiddroplets 25 Solution Step4 Exclusionofremainingstromalcontaminants Asmallprobabilityevent Somestromalcellsmayadheretothefreelipiddroplets Theremainingstromalcontaminantsbearingthefluorescentantibodies phycoerythrin PE conjugated tostromallineagemarkersarestainedtoexclude Negativeselection