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)前第1頁\共有28頁\編于星期四\9點(diǎn)2CONTENTSBackgroundAimoftheresearchDifficultySolution當(dāng)前第2頁\共有28頁\編于星期四\9點(diǎn)BackgroundWhatareadipocytes

(fatcells)?

Majorcellsintheadiposetissue.3當(dāng)前第3頁\共有28頁\編于星期四\9點(diǎn)BackgroundLipiddropletTwokindsofadipocytesinhuman4當(dāng)前第4頁\共有28頁\編于星期四\9點(diǎn)Prospectsofadipocytebiology

BackgroundLipidstoreInhibiteatingIncreasemetabolismLeptin5當(dāng)前第5頁\共有28頁\編于星期四\9點(diǎn)Prospectsofadipocytebiology

BackgroundAdipokinesInvolvedinenergymetabolismLipidstore6當(dāng)前第6頁\共有28頁\編于星期四\9點(diǎn)Prospectsofadipocytebiology

Specificallyfromadipose

tissue

BackgroundLipidstoreAdipokinesEnergymetabolism7當(dāng)前第7頁\共有28頁\編于星期四\9點(diǎn)Prospectsofadipocytebiology

Adipokinesplayansignificantroleinbiologicalfunction.

Background8當(dāng)前第8頁\共有28頁\編于星期四\9點(diǎn)BackgroundFlowCytometry(FCM)Principle

diagram9當(dāng)前第9頁\共有28頁\編于星期四\9點(diǎn)BackgroundVolume,Morphologicalcomplexityofcells,

TotalDNA/RNAcontent,Apoptosis,Cellviability,

......

......10當(dāng)前第10頁\共有28頁\編于星期四\9點(diǎn)AimoftheresearchAmethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.AnalysisFlowcytometry(FCM)Confocallaserscanningmicroscope(CLSM)Isolation

Fluorescence-activatedcellsorting(FACS)11當(dāng)前第11頁\共有28頁\編于星期四\9點(diǎn)AimoftheresearchAnalysisFlowcytometry(FCM)Confocallaserscanningmicroscope(CLSM)BeckmanCoulter--MoFlo?XDPAnalysisrate:100,000events/sec.Sortrate:70,000events/sec.12Amethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.當(dāng)前第12頁\共有28頁\編于星期四\9點(diǎn)AimoftheresearchAnalysisFlowcytometry(FCM)Confocallaserscanningmicroscope(CLSM)QuantitiveHighresolutionimaging13Amethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.當(dāng)前第13頁\共有28頁\編于星期四\9點(diǎn)AimoftheresearchAnalysisFlowcytometry(FCM)Confocallaserscanningmicroscope(CLSM)ImagingFlowCytometer(Amnis)

12ImagesforeverycellQuantitiveHighresolutionimaging14Amethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.當(dāng)前第14頁\共有28頁\編于星期四\9點(diǎn)AimoftheresearchIsolation

Fluorescence-activatedcellsorting(FACS)Apassingcellcanbeselectivelyadddifferentcharge,fallingintodifferentcontainers.15Amethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.當(dāng)前第15頁\共有28頁\編于星期四\9點(diǎn)DifficultyAdipocytesarelargeandfragile50–200μmEasytobebroken,whentheflowrate(pressure)islarge.16當(dāng)前第16頁\共有28頁\編于星期四\9點(diǎn)DifficultyAdipocytesarelargeandfragile50–200μmEasytobebroken,whentheflowrate(pressure)islarge.ContaminantsStromal/vascularcellsAggregatesofadipocytesFreelipiddropletsOtherdebris17當(dāng)前第17頁\共有28頁\編于星期四\9點(diǎn)SolutionAdipocytesarelargeandfragile50–200μm——usingflowcytometrywithlargerinternaldiameterofthefluidics(150to250μm).Easytobebroken——controlFlowpressuresto5~10psi,slowbutnotbroken.Goodforisolation.18當(dāng)前第18頁\共有28頁\編于星期四\9點(diǎn)SolutionContaminantsStromal/vascularcellsAggregatesofadipocytesFreelipiddropletsOtherdebris

——ExclusionofContaminants(4steps)19當(dāng)前第19頁\共有28頁\編于星期四\9點(diǎn)SolutionCollagenase——DigestionFiltration——Undigestedtissuefragmentsareexcluded.Centrifugation——Separatedfromstromal/vascularcellsFragile——UserockingratherthanvortexingPreparationofsinglecellsuspensionoffree-floatingadipocytes20當(dāng)前第20頁\共有28頁\編于星期四\9點(diǎn)SolutionStep1.Initialseparation——bysizeAdipocytes(50–200μm)biggerthanstromal/vascularcells(<20μm)—higherinForwardScatter(FSC)Thepresenceofhighlyrefractilelipiddropletsinadipocyte—higherinSSCOverlap——needfurtherseparationSmalladipocytes——ignored

(entireanalyzeifnecessary)21當(dāng)前第21頁\共有28頁\編于星期四\9點(diǎn)SolutionContaminantsStromal/vascularcellsAggregatesofadipocytesFreelipiddropletsOtherdebris——ExclusionofContaminants(4steps)22當(dāng)前第22頁\共有28頁\編于星期四\9點(diǎn)SolutionStep2.ExclusionofaggregatesandselectionofsingletsAggregatescanbeidentifiedbydotplot(WidthorArea).3parameters:Height

—voltage,Width—passingtime(size),Area=HxWWhenaggregates(doublets)pass,spendmoretime,Wincreased23當(dāng)前第23頁\共有28頁\編于星期四\9點(diǎn)SolutionStep2.ExclusionofaggregatesandselectionofsingletsStainnucleibyDyeCycleViolet(DCV)fluorescence.

Singlecellscanbeeasilygatedfromaggregates&otherdebrisThisstepcouldn’texcluderemainingstromal/vascularcells.24當(dāng)前第24頁\共有28頁\編于星期四\9點(diǎn)SolutionStep3.Exclusionofsinglestromal/vascularcellsStainthelipiddropletsbyLipidTOXDeepRed(LpdTX)

PositiveSelectionofeventscontaininglipiddroplets25當(dāng)前第25頁\共有28頁\編于星期四\9點(diǎn)SolutionStep4:ExclusionofremainingstromalcontaminantsAsmallprobabilityevent:Somestromalcellsmayadheretothefreelipiddroplets.Theremainingstromalcontaminantsbearingthefluorescentantibodies(phycoerythrin(PE)-conjugated)tostromallineagemarkersarestainedtoexclude.(Negativeselection)Negativeselection26當(dāng)前