用流式細(xì)胞儀分析脂肪細(xì)胞

AnalysisandIsolationofAdipocytesbyFlowCytometryYangLI2016-04-05MajkaSM,MillerHL,HelmKM,etal.AnalysisandIsolationofAdipocytesbyFlowCytometry.Methodsinenzymology.2014;537:281-296.doi:10.1016/B978-0-12-411619-1.00015-X.當(dāng)前1頁，共28頁，星期日。2CONTENTSBackgroundAimoftheresearchDifficultySolution當(dāng)前2頁，共28頁，星期日。BackgroundWhatareadipocytes

(fatcells)?

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

BackgroundLipidstoreInhibiteatingIncreasemetabolismLeptin5當(dāng)前5頁，共28頁，星期日。Prospectsofadipocytebiology

BackgroundAdipokinesInvolvedinenergymetabolismLipidstore6當(dāng)前6頁，共28頁，星期日。Prospectsofadipocytebiology

Specificallyfromadipose

tissue

BackgroundLipidstoreAdipokinesEnergymetabolism7當(dāng)前7頁，共28頁，星期日。Prospectsofadipocytebiology

Adipokinesplayansignificantroleinbiologicalfunction.

Background8當(dāng)前8頁，共28頁，星期日。BackgroundFlowCytometry(FCM)Principle

diagram9當(dāng)前9頁，共28頁，星期日。BackgroundVolume,Morphologicalcomplexityofcells,

TotalDNA/RNAcontent,Apoptosis,Cellviability,

......

......10當(dāng)前10頁，共28頁，星期日。AimoftheresearchAmethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.AnalysisFlowcytometry(FCM)Confocallaserscanningmicroscope(CLSM)Isolation

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

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

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

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

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

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

Singlecellscanbeeasilygatedfromaggregates&otherdebrisThisstepcouldn’texcluderemainingstromal/vascularcells.24當(dāng)前24頁，共28頁，星期日。SolutionStep3.Exclusionofsinglestromal/vascularcellsStainthelipiddropletsbyLipidTOXDee