AnalysisandIsolationofAdipocytesbyFlowCytometryYangLI2016-04-05MajkaSM,MillerHL,HelmKM,etal.AnalysisandIsolationofAdipocytesbyFlowCytometry.Methodsinenzymology.2014;537:281-296.doi:10.1016/B978-0-12-411619-1.00015-X.本文檔共28頁；當前第1頁；編輯于星期一\17點49分2CONTENTSBackgroundAimoftheresearchDifficultySolution本文檔共28頁；當前第2頁；編輯于星期一\17點49分BackgroundWhatareadipocytes

(fatcells)?

Majorcellsintheadiposetissue.3本文檔共28頁；當前第3頁；編輯于星期一\17點49分BackgroundLipiddropletTwokindsofadipocytesinhuman4本文檔共28頁；當前第4頁；編輯于星期一\17點49分Prospectsofadipocytebiology

BackgroundLipidstoreInhibiteatingIncreasemetabolismLeptin5本文檔共28頁；當前第5頁；編輯于星期一\17點49分Prospectsofadipocytebiology

BackgroundAdipokinesInvolvedinenergymetabolismLipidstore6本文檔共28頁；當前第6頁；編輯于星期一\17點49分Prospectsofadipocytebiology

Specificallyfromadipose

tissue

BackgroundLipidstoreAdipokinesEnergymetabolism7本文檔共28頁；當前第7頁；編輯于星期一\17點49分Prospectsofadipocytebiology

Adipokinesplayansignificantroleinbiologicalfunction.

Background8本文檔共28頁；當前第8頁；編輯于星期一\17點49分BackgroundFlowCytometry(FCM)Principle

diagram9本文檔共28頁；當前第9頁；編輯于星期一\17點49分BackgroundVolume,Morphologicalcomplexityofcells,

TotalDNA/RNAcontent,Apoptosis,Cellviability,

......

......10本文檔共28頁；當前第10頁；編輯于星期一\17點49分AimoftheresearchAmethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.AnalysisFlowcytometry(FCM)Confocallaserscanningmicroscope(CLSM)Isolation

Fluorescence-activatedcellsorting(FACS)11本文檔共28頁；當前第11頁；編輯于星期一\17點49分AimoftheresearchAnalysisFlowcytometry(FCM)Confocallaserscanningmicroscope(CLSM)BeckmanCoulter--MoFlo?XDPAnalysisrate:100,000events/sec.Sortrate:70,000events/sec.12Amethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.本文檔共28頁；當前第12頁；編輯于星期一\17點49分AimoftheresearchAnalysisFlowcytometry(FCM)Confocallaserscanningmicroscope(CLSM)QuantitiveHighresolutionimaging13Amethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.本文檔共28頁；當前第13頁；編輯于星期一\17點49分AimoftheresearchAnalysisFlowcytometry(FCM)Confocallaserscanningmicroscope(CLSM)ImagingFlowCytometer(Amnis)

12ImagesforeverycellQuantitiveHighresolutionimaging14Amethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.本文檔共28頁；當前第14頁；編輯于星期一\17點49分AimoftheresearchIsolation

Fluorescence-activatedcellsorting(FACS)Apassingcellcanbeselectivelyadddifferentcharge,fallingintodifferentcontainers.15Amethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.本文檔共28頁；當前第15頁；編輯于星期一\17點49分DifficultyAdipocytesarelargeandfragile50–200μmEasytobebroken,whentheflowrate(pressure)islarge.16本文檔共28頁；當前第16頁；編輯于星期一\17點49分DifficultyAdipocytesarelargeandfragile50–200μmEasytobebroken,whentheflowrate(pressure)islarge.ContaminantsStromal/vascularcellsAggregatesofadipocytesFreelipiddropletsOtherdebris17本文檔共28頁；當前第17頁；編輯于星期一\17點49分SolutionAdipocytesarelargeandfragile50–200μm——usingflowcytometrywithlargerinternaldiameterofthefluidics(150to250μm).Easytobebroken——controlFlowpressuresto5~10psi,slowbutnotbroken.Goodforisolation.18本文檔共28頁；當前第18頁；編輯于星期一\17點49分SolutionContaminantsStromal/vascularcellsAggregatesofadipocytesFreelipiddropletsOtherdebris

——ExclusionofContaminants(4steps)19本文檔共28頁；當前第19頁；編輯于星期一\17點49分SolutionCollagenase——DigestionFiltration——Undigestedtissuefragmentsareexcluded.Centrifugation——Separatedfromstromal/vascularcellsFragile——UserockingratherthanvortexingPreparationofsinglecellsuspensionoffree-floatingadipocytes20本文檔共28頁；當前第20頁；編輯于星期一\17點49分SolutionStep1.Initialseparation——bysizeAdipocytes(50–200μm)biggerthanstromal/vascularcells(<20μm)—higherinForwardScatter(FSC)Thepresenceofhighlyrefractilelipiddropletsinadipocyte—higherinSSCOverlap——needfurtherseparationSmalladipocytes——ignored

(entireanalyzeifnecessary)21本文檔共28頁；當前第21頁；編輯于星期一\17點49分SolutionContaminantsStromal/vascularcellsAggregatesofadipocytesFreelipiddropletsOtherdebris——ExclusionofContaminants(4steps)22本文檔共28頁；當前第22頁；編輯于星期一\17點49分SolutionStep2.ExclusionofaggregatesandselectionofsingletsAggregatescanbeidentifiedbydotplot(WidthorArea).3parameters:

Height

—voltage,Width—passingtime(size),Area=HxWWhenaggregates(doublets)pass,spendmoretime,Wincreased23本文檔共28頁；當前第23頁；編輯于星期一\17點49分SolutionStep2.ExclusionofaggregatesandselectionofsingletsStainnucleibyDyeCycleViolet(DCV)fluorescence.

Singlecellscanbeeasilygatedfromaggregates&otherdebrisThisstepcouldn’texcluderemainingstromal/vascularcells.24本文檔共28頁；當前第24頁；編輯于星期一\17點49分SolutionStep3.Exclusionofsinglestromal/vascularcellsStainthelipiddropletsbyLipidTOXDeepRed(LpdTX)

PositiveSelectionofeventscontaininglipiddroplets25本文檔共28頁；當前第25頁；編輯于星期一\17點49分SolutionStep4:Exclusionofremainingstromalcontaminants