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.当前1页，共28页，星期日。2CONTENTSBackgroundAimoftheresearchDifficultySolution当前2页，共28页，星期日。BackgroundWhatareadipocytes

(fatcells)?

Majorcellsintheadiposetissue.3当前3页，共28页，星期日。BackgroundLipiddropletTwokindsofadipocytesinhuman4当前4页，共28页，星期日。当前21页，共28页，星期日。Thisstepcouldn’texcluderemainingstromal/vascularcells.Filtration——Undigestedtissuefragmentsareexcluded.Negativeselection当前14页，共28页，星期日。Amethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.Stromal/vascularcellsThepresenceofhighlyrefractilelipiddropletsinadipocyte—higherinSSCAimoftheresearchAmethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.White--afterflowsorting(Almostundetectable)Flowcytometry(FCM)BeckmanCoulter--MoFlo™XDP当前1页，共28页，星期日。ExclusionofaggregatesandselectionofsingletsProspectsofadipocytebiology

BackgroundLipidstoreInhibiteatingIncreasemetabolismLeptin5当前5页，共28页，星期日。Prospectsofadipocytebiology

BackgroundAdipokinesInvolvedinenergymetabolismLipidstore6当前6页，共28页，星期日。Prospectsofadipocytebiology

Specificallyfromadipose

tissue

BackgroundLipidstoreAdipokinesEnergymetabolism7当前7页，共28页，星期日。Prospectsofadipocytebiology

Adipokinesplayansignificantroleinbiologicalfunction.

Background8当前8页，共28页，星期日。BackgroundFlowCytometry(FCM)Principle

diagram9当前9页，共28页，星期日。BackgroundVolume,Morphologicalcomplexityofcells,

TotalDNA/RNAcontent,Apoptosis,Cellviability,

......

......10当前10页，共28页，星期日。AimoftheresearchAmethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.AnalysisFlowcytometry(FCM)Confocallaserscanningmicroscope(CLSM)Isolation

Fluorescence-activatedcellsorting(FACS)11当前11页，共28页，星期日。AimoftheresearchAnalysisFlowcytometry(FCM)Confocallaserscanningmicroscope(CLSM)BeckmanCoulter--MoFlo™XDPAnalysisrate:100,000events/sec.Sortrate:70,000events/sec.12Amethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.当前12页，共28页，星期日。当前15页，共28页，星期日。ContaminantsStromal/vascularcellsImagingFlowCytometer(Amnis)当前26页，共28页，星期日。StainnucleibyDyeCycleViolet(DCV)fluorescence.ContaminantsFlowcytometry(FCM)ContaminantsProspectsofadipocytebiologyAmethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.AnalysisandIsolationofAdipocytesbyFlowCytometry.当前22页，共28页，星期日。AdipocytesarelargeandfragileConfocallaserscanningmicroscope(CLSM)AimoftheresearchAnalysisFlowcytometry(FCM)Confocallaserscanningmicroscope(CLSM)QuantitiveHighresolutionimaging13Amethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.当前13页，共28页，星期日。AimoftheresearchAnalysisFlowcytometry(FCM)Confocallaserscanningmicroscope(CLSM)ImagingFlowCytometer(Amnis)12ImagesforeverycellQuantitiveHighresolutionimaging14Amethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.当前14页，共28页，星期日。AimoftheresearchIsolation

Fluorescence-activatedcellsorting(FACS)Apassingcellcanbeselectivelyadddifferentcharge,fallingintodifferentcontainers.15Amethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.当前15页，共28页，星期日。DifficultyAdipocytesarelargeandfragile50–200μmEasytobebroken,whentheflowrate(pressure)islarge.16当前16页，共28页，星期日。DifficultyAdipocytesarelargeandfragile50–200μmEasytobebroken,whentheflowrate(pressure)islarge.ContaminantsStromal/vascularcellsAggregatesofadipocytesFreelipiddropletsOtherdebris17当前17页，共28页，星期日。SolutionAdipocytesarelargeandfragile50–200μm——usingflowcytometrywithlargerinternaldiameterofthefluidics(150to250μm).Easytobebroken——controlFlowpressuresto5~10psi,slowbutnotbroken.Goodforisolation.18当前18页，共28页，星期日。SolutionContaminantsStromal/vascularcellsAggregatesofadipocytesFreelipiddropletsOtherdebris

——ExclusionofContaminants(4steps)19当前19页，共28页，星期日。SolutionCollagenase——DigestionFiltration——Undigestedtissuefragmentsareexcluded.Centrifugation——Separatedfromstromal/vascularcellsFragile——UserockingratherthanvortexingPreparationofsinglecellsuspensionoffree-floatingadipocytes20当前20页，共28页，星期日。SolutionStep1.Initialseparation——bysizeAdipocytes(50–200μm)biggerthanstromal/vascularcells(<20μm)—higherinForwardScatter(FSC)Thepresenceofhighlyrefractilelipiddropletsinadipocyte—higherinSSCOverlap——needfurtherseparationSmalladipocytes——ignored

(entireanalyzeifnecessary)21当前21页，共28页，星期日。当前19页，共28页，星期日。Sortrate:70,000events/sec.2014;537:281-296.当前27页，共28页，星期日。IncreasemetabolismFiltration——Undigestedtissuefragmentsareexcluded.PrinciplediagramFlowcytometry(FCM)当前14页，共28页，星期日。Whatareadipocytes(fatcells)?

Majorcellsintheadiposetissue.Exclusionofaggregatesandselectionofsinglets当前15页，共28页，星期日。Whenaggregates(doublets)pass,spendmoretime,WincreasedNegativeselectionAimoftheresearchSolutionContaminantsStromal/vascularcellsAggregatesofadipocytesFreelipiddropletsOtherdebris——ExclusionofContaminants(4steps)22当前22页，共28页，星期日。SolutionStep2.ExclusionofaggregatesandselectionofsingletsAggregatescanbeidentifiedbydotplot(WidthorArea).3parameters:Height

—voltage,Width—passingtime(size),Area=HxWWhenaggregates(doublets)pass,spendmoretime,Wincreased23当前23页，共28页，星期日。SolutionStep2.ExclusionofaggregatesandselectionofsingletsStainnucleibyDyeCycleViolet(DCV)fluorescence.

Singlecellscanbeeasilygatedfromaggregates&otherdebrisThisstepcouldn’texcluderemainingstromal/vascularcells.24当前24页，共28页，星期日。SolutionStep3.Exclusionofsinglestromal/vascularcellsStainthelipiddropletsbyLipidTOXDee