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.現(xiàn)在是1頁(yè)\一共有28頁(yè)\編輯于星期二2CONTENTSBackgroundAimoftheresearchDifficultySolution現(xiàn)在是2頁(yè)\一共有28頁(yè)\編輯于星期二BackgroundWhatareadipocytes

(fatcells)?

Majorcellsintheadiposetissue.3現(xiàn)在是3頁(yè)\一共有28頁(yè)\編輯于星期二BackgroundLipiddropletTwokindsofadipocytesinhuman4現(xiàn)在是4頁(yè)\一共有28頁(yè)\編輯于星期二Prospectsofadipocytebiology

BackgroundLipidstoreInhibiteatingIncreasemetabolismLeptin5現(xiàn)在是5頁(yè)\一共有28頁(yè)\編輯于星期二Prospectsofadipocytebiology

BackgroundAdipokinesInvolvedinenergymetabolismLipidstore6現(xiàn)在是6頁(yè)\一共有28頁(yè)\編輯于星期二Prospectsofadipocytebiology

Specificallyfromadipose

tissue

BackgroundLipidstoreAdipokinesEnergymetabolism7現(xiàn)在是7頁(yè)\一共有28頁(yè)\編輯于星期二Prospectsofadipocytebiology

Adipokinesplayansignificantroleinbiologicalfunction.

Background8現(xiàn)在是8頁(yè)\一共有28頁(yè)\編輯于星期二BackgroundFlowCytometry(FCM)Principle

diagram9現(xiàn)在是9頁(yè)\一共有28頁(yè)\編輯于星期二BackgroundVolume,Morphologicalcomplexityofcells,

TotalDNA/RNAcontent,Apoptosis,Cellviability,

......

......10現(xiàn)在是10頁(yè)\一共有28頁(yè)\編輯于星期二AimoftheresearchAmethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.AnalysisFlowcytometry(FCM)Confocallaserscanningmicroscope(CLSM)Isolation

Fluorescence-activatedcellsorting(FACS)11現(xiàn)在是11頁(yè)\一共有28頁(yè)\編輯于星期二AimoftheresearchAnalysisFlowcytometry(FCM)Confocallaserscanningmicroscope(CLSM)BeckmanCoulter--MoFlo?XDPAnalysisrate:100,000events/sec.Sortrate:70,000events/sec.12Amethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.現(xiàn)在是12頁(yè)\一共有28頁(yè)\編輯于星期二AimoftheresearchAnalysisFlowcytometry(FCM)Confocallaserscanningmicroscope(CLSM)QuantitiveHighresolutionimaging13Amethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.現(xiàn)在是13頁(yè)\一共有28頁(yè)\編輯于星期二AimoftheresearchAnalysisFlowcytometry(FCM)Confocallaserscanningmicroscope(CLSM)ImagingFlowCytometer(Amnis)

12ImagesforeverycellQuantitiveHighresolutionimaging14Amethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.現(xiàn)在是14頁(yè)\一共有28頁(yè)\編輯于星期二AimoftheresearchIsolation

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

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

(entireanalyzeifnecessary)21現(xiàn)在是21頁(yè)\一共有28頁(yè)\編輯于星期二SolutionContaminantsStromal/vascularcellsAggregatesofadipocytesFreelipiddropletsOtherdebris——ExclusionofContaminants(4steps)22現(xiàn)在是22頁(yè)\一共有28頁(yè)\編輯于星期二SolutionStep2.ExclusionofaggregatesandselectionofsingletsAggregatescanbeidentifiedbydotplot(WidthorArea).3parameters:

Height

—voltage,Width—passingtime(size),Area=HxWWhenaggregates(doublets)pass,spendmoretime,Wincreased23現(xiàn)在是23頁(yè)\一共有28頁(yè)\編輯于星期二SolutionStep2.ExclusionofaggregatesandselectionofsingletsStainnucleibyDyeCycleViolet(DCV)fluorescence.

Singlecellscanbeeasilygatedfromaggregates&otherdebrisThisstepcouldn’texcluderemainingstromal/vascularcells.24現(xiàn)在是24頁(yè)\一共有28頁(yè)\編輯于星期二SolutionStep3.Exclusionofsinglestromal/vascularcellsStainthelipiddropletsbyLipidTOXDeepRed(LpdTX)

PositiveSelectionofeventscontaininglipiddroplets25現(xiàn)在是25頁(yè)\一共有28頁(yè)\編輯于星期二SolutionStep4:ExclusionofremainingstromalcontaminantsAsmallprobabilityevent:Somestromalcellsmayadheretothefreelipiddroplets.Theremainingstromalcontaminantsbearingthefluorescentantibodies(phycoerythrin(PE)-conjugated)tostromallineagemarkersarestainedtoexclude.(Negativeselection)Negativeselection26