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.现在是1页\一共有28页\编辑于星期二2CONTENTSBackgroundAimoftheresearchDifficultySolution现在是2页\一共有28页\编辑于星期二BackgroundWhatareadipocytes

(fatcells)?

Majorcellsintheadiposetissue.3现在是3页\一共有28页\编辑于星期二BackgroundLipiddropletTwokindsofadipocytesinhuman4现在是4页\一共有28页\编辑于星期二Prospectsofadipocytebiology

BackgroundLipidstoreInhibiteatingIncreasemetabolismLeptin5现在是5页\一共有28页\编辑于星期二Prospectsofadipocytebiology

BackgroundAdipokinesInvolvedinenergymetabolismLipidstore6现在是6页\一共有28页\编辑于星期二Prospectsofadipocytebiology

Specificallyfromadipose

tissue

BackgroundLipidstoreAdipokinesEnergymetabolism7现在是7页\一共有28页\编辑于星期二Prospectsofadipocytebiology

Adipokinesplayansignificantroleinbiologicalfunction.

Background8现在是8页\一共有28页\编辑于星期二BackgroundFlowCytometry(FCM)Principle

diagram9现在是9页\一共有28页\编辑于星期二BackgroundVolume,Morphologicalcomplexityofcells,

TotalDNA/RNAcontent,Apoptosis,Cellviability,

......

......10现在是10页\一共有28页\编辑于星期二AimoftheresearchAmethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.AnalysisFlowcytometry(FCM)Confocallaserscanningmicroscope(CLSM)Isolation

Fluorescence-activatedcellsorting(FACS)11现在是11页\一共有28页\编辑于星期二AimoftheresearchAnalysisFlowcytometry(FCM)Confocallaserscanningmicroscope(CLSM)BeckmanCoulter--MoFlo™XDPAnalysisrate:100,000events/sec.Sortrate:70,000events/sec.12Amethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.现在是12页\一共有28页\编辑于星期二AimoftheresearchAnalysisFlowcytometry(FCM)Confocallaserscanningmicroscope(CLSM)QuantitiveHighresolutionimaging13Amethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.现在是13页\一共有28页\编辑于星期二AimoftheresearchAnalysisFlowcytometry(FCM)Confocallaserscanningmicroscope(CLSM)ImagingFlowCytometer(Amnis)

12ImagesforeverycellQuantitiveHighresolutionimaging14Amethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.现在是14页\一共有28页\编辑于星期二AimoftheresearchIsolation

Fluorescence-activatedcellsorting(FACS)Apassingcellcanbeselectivelyadddifferentcharge,fallingintodifferentcontainers.15Amethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.现在是15页\一共有28页\编辑于星期二DifficultyAdipocytesarelargeandfragile50–200μmEasytobebroken,whentheflowrate(pressure)islarge.16现在是16页\一共有28页\编辑于星期二DifficultyAdipocytesarelargeandfragile50–200μmEasytobebroken,whentheflowrate(pressure)islarge.ContaminantsStromal/vascularcellsAggregatesofadipocytesFreelipiddropletsOtherdebris17现在是17页\一共有28页\编辑于星期二SolutionAdipocytesarelargeandfragile50–200μm——usingflowcytometrywithlargerinternaldiameterofthefluidics(150to250μm).Easytobebroken——controlFlowpressuresto5~10psi,slowbutnotbroken.Goodforisolation.18现在是18页\一共有28页\编辑于星期二SolutionContaminantsStromal/vascularcellsAggregatesofadipocytesFreelipiddropletsOtherdebris

——ExclusionofContaminants(4steps)19现在是19页\一共有28页\编辑于星期二SolutionCollagenase——DigestionFiltration——Undigestedtissuefragmentsareexcluded.Centrifugation——Separatedfromstromal/vascularcellsFragile——UserockingratherthanvortexingPreparationofsinglecellsuspensionoffree-floatingadipocytes20现在是20页\一共有28页\编辑于星期二SolutionStep1.Initialseparation——bysizeAdipocytes(50–200μm)biggerthanstromal/vascularcells(<20μm)—higherinForwardScatter(FSC)Thepresenceofhighlyrefractilelipiddropletsinadipocyte—higherinSSCOverlap——needfurtherseparationSmalladipocytes——ignored

(entireanalyzeifnecessary)21现在是21页\一共有28页\编辑于星期二SolutionContaminantsStromal/vascularcellsAggregatesofadipocytesFreelipiddropletsOtherdebris——ExclusionofContaminants(4steps)22现在是22页\一共有28页\编辑于星期二SolutionStep2.ExclusionofaggregatesandselectionofsingletsAggregatescanbeidentifiedbydotplot(WidthorArea).3parameters:

Height

—voltage,Width—passingtime(size),Area=HxWWhenaggregates(doublets)pass,spendmoretime,Wincreased23现在是23页\一共有28页\编辑于星期二SolutionStep2.ExclusionofaggregatesandselectionofsingletsStainnucleibyDyeCycleViolet(DCV)fluorescence.

Singlecellscanbeeasilygatedfromaggregates&otherdebrisThisstepcouldn’texcluderemainingstromal/vascularcells.24现在是24页\一共有28页\编辑于星期二SolutionStep3.Exclusionofsinglestromal/vascularcellsStainthelipiddropletsbyLipidTOXDeepRed(LpdTX)

PositiveSelectionofeventscontaininglipiddroplets25现在是25页\一共有28页\编辑于星期二SolutionStep4:ExclusionofremainingstromalcontaminantsAsmallprobabilityevent:Somestromalcellsmayadheretothefreelipiddroplets.Theremainingstromalcontaminantsbearingthefluorescentantibodies(phycoerythrin(PE)-conjugated)tostromallineagemarkersarestainedtoexclude.(Negativeselection)Negativeselection26