用流式细胞仪分析脂肪细胞.pptx

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1、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.2CONTENTSBackgroundAim of the researchDifficulty SolutionBackgroundWhat are

2、 adipocytes(fat cells)?Majorcellsintheadiposetissue.3BackgroundLipid dropletTwokindsofadipocytesinhuman4ProspectsofadipocytebiologyBackgroundLipidstoreInhibiteatingIncreasemetabolismLeptin5ProspectsofadipocytebiologyBackgroundAdipokinesInvolvedinenergymetabolismLipidstore6Prospectsofadipocytebiology

3、SpecificallyfromadiposetissueBackgroundLipidstoreAdipokinesEnergymetabolism7ProspectsofadipocytebiologyAdipokinesplayansignificantroleinbiologicalfunction.Background8BackgroundFlowCytometry(FCM)Principlediagram9BackgroundVolume,Morphological complexity of cells,Total DNA/RNA content,Apoptosis,Cell v

4、iability,.10Aim of the researchAmethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.AnalysisFlow cytometry(FCM)Confocal laser scanning microscope(CLSM)IsolationFluorescence-activated cell sorting(FACS)11Aim of the researchAnalysisFlow cytometry(FCM)Confocal laser scanning microscope(

5、CLSM)Beckman Coulter-MoFlo XDPAnalysisrate:100,000events/sec.Sortrate:70,000events/sec.12Amethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.Aim of the researchAnalysisFlow cytometry(FCM)Confocal laser scanning microscope(CLSM)QuantitiveHighresolutionimaging13Amethodforanalysisandis

6、olationfree-floatingadipocytesbyFlowCytometry.Aim of the researchAnalysisFlow cytometry(FCM)Confocal laser scanning microscope(CLSM)Imaging Flow Cytometer(Amnis)12ImagesforeverycellQuantitiveHighresolutionimaging14Amethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.Aim of the resear

7、chIsolationFluorescence-activated cell sorting(FACS)A passing cell can be selectively add different charge,falling into different containers.15Amethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.DifficultyAdipocytes are large and fragile 50200mEasytobebroken,whentheflowrate(pressure

8、)islarge.16DifficultyAdipocytes are large and fragile 50200mEasytobebroken,whentheflowrate(pressure)islarge.Contaminants Stromal/vascularcellsAggregatesofadipocytesFreelipiddropletsOtherdebris17SolutionAdipocytes are large and fragile 50200musingflowcytometrywithlargerinternaldiameterofthefluidics(1

9、50to250m).EasytobebrokencontrolFlowpressuresto510psi,slowbutnotbroken.Goodforisolation.18SolutionContaminants Stromal/vascularcellsAggregatesofadipocytesFreelipiddropletsOtherdebrisExclusionofContaminants(4steps)19SolutionCollagenase DigestionFiltration Undigested tissue fragments are excluded.Centr

10、ifugation Separated from stromal/vascular cellsFragile Use rocking rather than vortexingPreparation of single cell suspension of free-floating adipocytes20SolutionStep 1.Initial separationby size Adipocytes(50200m)biggerthanstromal/vascularcells(20m)higherinForwardScatter(FSC)Thepresenceofhighlyrefr

11、actilelipiddropletsinadipocytehigherinSSCOverlapneedfurtherseparationSmalladipocytesignored(entireanalyzeifnecessary)21SolutionContaminants Stromal/vascularcellsAggregatesofadipocytesFreelipiddropletsOtherdebrisExclusionofContaminants(4steps)22SolutionStep 2.Exclusion of aggregates and selection of

12、singlets Aggregatescanbeidentifiedbydotplot(WidthorArea).3parameters:Heightvoltage,Widthpassingtime(size),Area=HxWWhenaggregates(doublets)pass,spendmoretime,Wincreased23SolutionStep 2.Exclusion of aggregates and selection of singlets StainnucleibyDyeCycleViolet(DCV)fluorescence.Singlecellscanbeeasil

13、ygatedfromaggregates&otherdebrisThisstepcouldntexcluderemainingstromal/vascularcells.24SolutionStep 3.Exclusion of single stromal/vascular cells StainthelipiddropletsbyLipidTOXDeepRed(LpdTX)PositiveSelectionofeventscontaininglipiddroplets25SolutionStep 4:Exclusion of remaining stromal contaminants A

14、smallprobabilityevent:Somestromalcellsmayadheretothefreelipiddroplets.Theremainingstromalcontaminantsbearingthefluorescentantibodies(phycoerythrin(PE)-conjugated)tostromallineagemarkersarestainedtoexclude.(Negativeselection)Negativeselection26SolutionValidationqRT-PCRStromalcellsmarker(suchasCD34)Black-beforeflowsorting(Highexpressed)White-afterflowsorting(Almostundetectable)27Analysis and Isolation of Adipocytes by Flow CytometryYang LI