用流式细胞仪分析脂肪细胞

AnalysisandIsolationofAdipocytesbyFlowCytometryYangLI2016-04-05MajkaSM,MillerHL,HelmKM,etal.AnalysisandIsolationofAdipocytesbyFlowCytometry.Methodsinenzymology.2014;537:281-296.doi:10.1016/B978-0-12-411619-1.00015-X.本文档共28页；当前第1页；编辑于星期一\17点49分2CONTENTSBackgroundAimoftheresearchDifficultySolution本文档共28页；当前第2页；编辑于星期一\17点49分BackgroundWhatareadipocytes

(fatcells)?

Majorcellsintheadiposetissue.3本文档共28页；当前第3页；编辑于星期一\17点49分BackgroundLipiddropletTwokindsofadipocytesinhuman4本文档共28页；当前第4页；编辑于星期一\17点49分Prospectsofadipocytebiology

BackgroundLipidstoreInhibiteatingIncreasemetabolismLeptin5本文档共28页；当前第5页；编辑于星期一\17点49分Prospectsofadipocytebiology

BackgroundAdipokinesInvolvedinenergymetabolismLipidstore6本文档共28页；当前第6页；编辑于星期一\17点49分Prospectsofadipocytebiology

Specificallyfromadipose

tissue

BackgroundLipidstoreAdipokinesEnergymetabolism7本文档共28页；当前第7页；编辑于星期一\17点49分Prospectsofadipocytebiology

Adipokinesplayansignificantroleinbiologicalfunction.

Background8本文档共28页；当前第8页；编辑于星期一\17点49分BackgroundFlowCytometry(FCM)Principle

diagram9本文档共28页；当前第9页；编辑于星期一\17点49分BackgroundVolume,Morphologicalcomplexityofcells,

TotalDNA/RNAcontent,Apoptosis,Cellviability,

......

......10本文档共28页；当前第10页；编辑于星期一\17点49分AimoftheresearchAmethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.AnalysisFlowcytometry(FCM)Confocallaserscanningmicroscope(CLSM)Isolation

Fluorescence-activatedcellsorting(FACS)11本文档共28页；当前第11页；编辑于星期一\17点49分AimoftheresearchAnalysisFlowcytometry(FCM)Confocallaserscanningmicroscope(CLSM)BeckmanCoulter--MoFlo™XDPAnalysisrate:100,000events/sec.Sortrate:70,000events/sec.12Amethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.本文档共28页；当前第12页；编辑于星期一\17点49分AimoftheresearchAnalysisFlowcytometry(FCM)Confocallaserscanningmicroscope(CLSM)QuantitiveHighresolutionimaging13Amethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.本文档共28页；当前第13页；编辑于星期一\17点49分AimoftheresearchAnalysisFlowcytometry(FCM)Confocallaserscanningmicroscope(CLSM)ImagingFlowCytometer(Amnis)

12ImagesforeverycellQuantitiveHighresolutionimaging14Amethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.本文档共28页；当前第14页；编辑于星期一\17点49分AimoftheresearchIsolation

Fluorescence-activatedcellsorting(FACS)Apassingcellcanbeselectivelyadddifferentcharge,fallingintodifferentcontainers.15Amethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.本文档共28页；当前第15页；编辑于星期一\17点49分DifficultyAdipocytesarelargeandfragile50–200μmEasytobebroken,whentheflowrate(pressure)islarge.16本文档共28页；当前第16页；编辑于星期一\17点49分DifficultyAdipocytesarelargeandfragile50–200μmEasytobebroken,whentheflowrate(pressure)islarge.ContaminantsStromal/vascularcellsAggregatesofadipocytesFreelipiddropletsOtherdebris17本文档共28页；当前第17页；编辑于星期一\17点49分SolutionAdipocytesarelargeandfragile50–200μm——usingflowcytometrywithlargerinternaldiameterofthefluidics(150to250μm).Easytobebroken——controlFlowpressuresto5~10psi,slowbutnotbroken.Goodforisolation.18本文档共28页；当前第18页；编辑于星期一\17点49分SolutionContaminantsStromal/vascularcellsAggregatesofadipocytesFreelipiddropletsOtherdebris

——ExclusionofContaminants(4steps)19本文档共28页；当前第19页；编辑于星期一\17点49分SolutionCollagenase——DigestionFiltration——Undigestedtissuefragmentsareexcluded.Centrifugation——Separatedfromstromal/vascularcellsFragile——UserockingratherthanvortexingPreparationofsinglecellsuspensionoffree-floatingadipocytes20本文档共28页；当前第20页；编辑于星期一\17点49分SolutionStep1.Initialseparation——bysizeAdipocytes(50–200μm)biggerthanstromal/vascularcells(<20μm)—higherinForwardScatter(FSC)Thepresenceofhighlyrefractilelipiddropletsinadipocyte—higherinSSCOverlap——needfurtherseparationSmalladipocytes——ignored

(entireanalyzeifnecessary)21本文档共28页；当前第21页；编辑于星期一\17点49分SolutionContaminantsStromal/vascularcellsAggregatesofadipocytesFreelipiddropletsOtherdebris——ExclusionofContaminants(4steps)22本文档共28页；当前第22页；编辑于星期一\17点49分SolutionStep2.ExclusionofaggregatesandselectionofsingletsAggregatescanbeidentifiedbydotplot(WidthorArea).3parameters:

Height

—voltage,Width—passingtime(size),Area=HxWWhenaggregates(doublets)pass,spendmoretime,Wincreased23本文档共28页；当前第23页；编辑于星期一\17点49分SolutionStep2.ExclusionofaggregatesandselectionofsingletsStainnucleibyDyeCycleViolet(DCV)fluorescence.

Singlecellscanbeeasilygatedfromaggregates&otherdebrisThisstepcouldn’texcluderemainingstromal/vascularcells.24本文档共28页；当前第24页；编辑于星期一\17点49分SolutionStep3.Exclusionofsinglestromal/vascularcellsStainthelipiddropletsbyLipidTOXDeepRed(LpdTX)

PositiveSelectionofeventscontaininglipiddroplets25本文档共28页；当前第25页；编辑于星期一\17点49分SolutionStep4:Exclusionofremainingstromalcontaminants