用流式细胞仪分析脂肪细胞

1、.,AnalysisandIsolationofAdipocytesbyFlowCytometry,YangLI2016-04-05,MajkaSM,MillerHL,HelmKM,etal.AnalysisandIsolationofAdipocytesbyFlowCytometry.Methodsinenzymology.2014;537:281-296.doi:10.1016/B978-0-12-411619-1.00015-X.,2,CONTENTS,BackgroundAimoftheresearchDifficultySolution,.,Background,Whatareadi

2、pocytes(fatcells)?Majorcellsintheadiposetissue.,3,.,Background,Lipiddroplet,Twokindsofadipocytesinhuman,4,.,Prospectsofadipocytebiology,Background,Lipidstore,InhibiteatingIncreasemetabolism,Leptin,5,.,Prospectsofadipocytebiology,Background,Adipokines,Involvedinenergymetabolism,Lipidstore,6,.,Prospec

3、tsofadipocytebiologySpecificallyfromadiposetissue,Background,Lipidstore,Adipokines,Energymetabolism,7,.,ProspectsofadipocytebiologyAdipokinesplayansignificantroleinbiologicalfunction.,Background,8,.,Background,FlowCytometry(FCM),Principlediagram,9,.,Background,Volume,Morphologicalcomplexityofcells,T

4、otalDNA/RNAcontent,Apoptosis,Cellviability,.,10,.,Aimoftheresearch,Amethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.,Analysis,Flowcytometry(FCM)Confocallaserscanningmicroscope(CLSM),Isolation,Fluorescence-activatedcellsorting(FACS),11,.,Aimoftheresearch,Analysis,Flowcytometry(FCM

5、)Confocallaserscanningmicroscope(CLSM),BeckmanCoulter-MoFloXDP,Analysisrate:100,000events/sec.Sortrate:70,000events/sec.,12,Amethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.,.,Aimoftheresearch,Analysis,Flowcytometry(FCM)Confocallaserscanningmicroscope(CLSM),Quantitive,Highresolut

6、ionimaging,13,Amethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.,.,Aimoftheresearch,Analysis,Flowcytometry(FCM)Confocallaserscanningmicroscope(CLSM),ImagingFlowCytometer(Amnis),12Imagesforeverycell,Quantitive,Highresolutionimaging,14,Amethodforanalysisandisolationfree-floatingadip

7、ocytesbyFlowCytometry.,.,Aimoftheresearch,Isolation,Fluorescence-activatedcellsorting(FACS),Apassingcellcanbeselectivelyadddifferentcharge,fallingintodifferentcontainers.,15,Amethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.,.,Difficulty,Adipocytesarelargeandfragile,50200mEasytobe

8、broken,whentheflowrate(pressure)islarge.,16,.,Difficulty,Adipocytesarelargeandfragile,50200mEasytobebroken,whentheflowrate(pressure)islarge.,Contaminants,Stromal/vascularcellsAggregatesofadipocytesFreelipiddropletsOtherdebris,17,.,Solution,Adipocytesarelargeandfragile,50200musingflowcytometrywithlar

9、gerinternaldiameterofthefluidics(150to250m).EasytobebrokencontrolFlowpressuresto510psi,slowbutnotbroken.Goodforisolation.,18,.,Solution,Contaminants,Stromal/vascularcellsAggregatesofadipocytesFreelipiddropletsOtherdebrisExclusionofContaminants(4steps),19,.,Solution,CollagenaseDigestionFiltrationUndi

10、gestedtissuefragmentsareexcluded.CentrifugationSeparatedfromstromal/vascularcellsFragileUserockingratherthanvortexing,Preparationofsinglecellsuspensionoffree-floatingadipocytes,20,.,Solution,Step1.Initialseparationbysize,Adipocytes(50200m)biggerthanstromal/vascularcells(20m)higherinForwardScatter(FS

11、C)ThepresenceofhighlyrefractilelipiddropletsinadipocytehigherinSSCOverlapneedfurtherseparationSmalladipocytesignored(entireanalyzeifnecessary),21,.,Solution,Contaminants,Stromal/vascularcellsAggregatesofadipocytesFreelipiddropletsOtherdebrisExclusionofContaminants(4steps),22,.,Solution,Step2.Exclusi

12、onofaggregatesandselectionofsinglets,Aggregatescanbeidentifiedbydotplot(WidthorArea).3parameters:Heightvoltage,Widthpassingtime(size),Area=HxWWhenaggregates(doublets)pass,spendmoretime,Wincreased,23,.,Solution,Step2.Exclusionofaggregatesandselectionofsinglets,StainnucleibyDyeCycleViolet(DCV)fluoresc

13、ence.Singlecellscanbeeasilygatedfromaggregates&otherdebrisThisstepcouldntexcluderemainingstromal/vascularcells.,24,.,Solution,Step3.Exclusionofsinglestromal/vascularcells,StainthelipiddropletsbyLipidTOXDeepRed(LpdTX)PositiveSelectionofeventscontaininglipiddroplets,25,.,Solution,Step4:Exclusionofremainingstromalcontaminants,Asmallprobabilityevent:Somestromalcellsmayadheretothefreelipiddroplets.Theremainingstromalcontaminantsbearingthefluorescentantibodies(phycoerythrin(PE)-conjugated)tostromallineagemarkersarestainedtoexclude.(Negativeselection),Neg