用流式细胞仪分析脂肪细胞.ppt

Analysis and Isolation of Adipocytes by Flow CytometryYang LI 2016-04-05MajkaSM,MillerHL,HelmKM,etal.AnalysisandIsolationofAdipocytesbyFlowCytometry.Methodsinenzymology.2014;537:281-296.doi:10.1016/B978-0-12-411619-1.00015-X.1.2024/5/22 周三2CONTENTSBackgroundAim of the researchDifficulty Solution2.2024/5/22 周三BackgroundWhat are adipocytes(fat cells)?Majorcellsintheadiposetissue.33.2024/5/22 周三BackgroundLipid dropletTwokindsofadipocytesinhuman44.2024/5/22 周三ProspectsofadipocytebiologyBackgroundLipidstoreInhibiteatingIncreasemetabolismLeptin55.2024/5/22 周三ProspectsofadipocytebiologyBackgroundAdipokinesInvolvedinenergymetabolismLipidstore66.2024/5/22 周三ProspectsofadipocytebiologySpecificallyfromadiposetissueBackgroundLipidstoreAdipokinesEnergymetabolism77.2024/5/22 周三ProspectsofadipocytebiologyAdipokinesplayansignificantroleinbiologicalfunction.Background88.2024/5/22 周三BackgroundFlowCytometry(FCM)Principlediagram99.2024/5/22 周三BackgroundVolume,Morphological complexity of cells,Total DNA/RNA content,Apoptosis,Cell viability,.1010.2024/5/22 周三Aim of the researchAmethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.AnalysisFlow cytometry(FCM)Confocal laser scanning microscope(CLSM)IsolationFluorescence-activated cell sorting(FACS)1111.2024/5/22 周三Aim of the researchAnalysisFlow cytometry(FCM)Confocal laser scanning microscope(CLSM)Beckman Coulter-MoFlo XDPAnalysisrate:100,000events/sec.Sortrate:70,000events/sec.12Amethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.12.2024/5/22 周三Aim of the researchAnalysisFlow cytometry(FCM)Confocal laser scanning microscope(CLSM)QuantitiveHighresolutionimaging13Amethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.13.2024/5/22 周三Aim of the researchAnalysisFlow cytometry(FCM)Confocal laser scanning microscope(CLSM)Imaging Flow Cytometer(Amnis)12ImagesforeverycellQuantitiveHighresolutionimaging14Amethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.14.2024/5/22 周三Aim of the researchIsolationFluorescence-activated cell sorting(FACS)A passing cell can be selectively add different charge,falling into different containers.15Amethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.15.2024/5/22 周三DifficultyAdipocytes are large and fragile 50200mEasytobebroken,whentheflowrate(pressure)islarge.1616.2024/5/22 周三DifficultyAdipocytes are large and fragile 50200mEasytobebroken,whentheflowrate(pressure)islarge.Contaminants Stromal/vascularcellsAggregatesofadipocytesFreelipiddropletsOtherdebris1717.2024/5/22 周三SolutionAdipocytes are large and fragile 50200musingflowcytometrywithlargerinternaldiameterofthefluidics(150to250m).EasytobebrokencontrolFlowpressuresto510psi,slowbutnotbroken.Goodforisolation.1818.2024/5/22 周三SolutionContaminants Stromal/vascularcellsAggregatesofadipocytesFreelipiddropletsOtherdebrisExclusionofContaminants(4steps)1919.2024/5/22 周三SolutionCollagenase DigestionFiltration Undigested tissue fragments are excluded.Centrifugation Separated from stromal/vascular cellsFragile Use rocking rather than vortexingPreparation of single cell suspension of free-floating adipocytes2020.2024/5/22 周三SolutionStep 1.Initial separationby size Adipocytes(50200m)biggerthanstromal/vascularcells(20m)higherinForwardScatter(FSC)ThepresenceofhighlyrefractilelipiddropletsinadipocytehigherinSSCOverlapneedfurtherseparationSmalladipocytesignored(entireanalyzeifnecessary)2121.2024/5/22 周三SolutionContaminants Stromal/vascularcellsAggregatesofadipocytesFreelipiddropletsOtherdebrisExclusionofContaminants(4steps)2222.2024/5/22 周三SolutionStep 2.Exclusion of aggregates and selection of singlets Aggregatescanbeidentifiedbydotplot(WidthorArea).3parameters:Heightvoltage,Widthpassingtime(size),Area=HxWWhenaggregates(doublets)pass,spendmoretime,Wincreased2323.2024/5/22 周三SolutionStep 2.Exclusion of aggregates and selection of singlets StainnucleibyDyeCycleViolet(DCV)fluorescence.Singlecellscanbeeasilygatedfromaggregates&otherdebrisThisstepcouldntexcluderemainingstromal/vascularcells.2424.2024/5/22 周三SolutionStep 3.Exclusion of single stromal/vascular cells StainthelipiddropletsbyLipidTOXDeepRed(LpdTX)PositiveSelectionofeventscontaininglipiddroplets2525.2024/5/22 周三SolutionStep 4:Exclusion of remaining stromal contaminants Asmallprobabilityevent:Somestromalcellsmayadheretothefreelipiddroplets.Theremainingstromalcontaminantsbearingthefluorescentantibodies(phycoerythrin(PE)-conjugated)tostromallineagemarkersarestainedtoexclude.(Negativeselection)Negativeselection2626.2024/5/22 周三SolutionValidationqRT-PCRStromalcellsmarker(suchasCD34)Black-beforeflowsorting(Highexpressed)White-afterflowsorting(Almostundetectable)2727.2024/5/22 周三Analysis and Isolation of Adipocytes by Flow CytometryYang LI 28.2024/5/22 周三