用流式细胞仪分析脂肪细胞专业课件

AnalysisandIsolationofAdipocytesbyFlowCytometryYangLI2021-04-05MajkaSM,MillerHL,HelmKM,etal.AnalysisandIsolationofAdipocytesbyFlowCytometry.Methodsinenzymology.2021;537:281-296.doi:10.1016/B978-0-12-411619-1.00015-X.12CONTENTSBackgroundAimoftheresearchDifficultySolution2BackgroundWhatareadipocytes

(fatcells)?

Majorcellsintheadiposetissue.33BackgroundLipiddropletTwokindsofadipocytesinhuman44Prospectsofadipocytebiology

BackgroundLipidstoreInhibiteatingIncreasemetabolismLeptin55Prospectsofadipocytebiology

BackgroundAdipokinesInvolvedinenergymetabolismLipidstore66Prospectsofadipocytebiology

Specificallyfromadipose

tissue

BackgroundLipidstoreAdipokinesEnergymetabolism77Prospectsofadipocytebiology

Adipokinesplayansignificantroleinbiologicalfunction.

Background88BackgroundFlowCytometry(FCM)Principle

diagram99BackgroundVolume,Morphologicalcomplexityofcells,

TotalDNA/RNAcontent,Apoptosis,Cellviability,

......

......10109、人的价值，在招收诱惑的一瞬间被决定。2023/2/32023/2/3Friday,February3,202310、低头要有勇气，抬头要有低气。2023/2/32023/2/32023/2/32/3/20234:58:04PM11、人总是珍惜为得到。2023/2/32023/2/32023/2/3Feb-2303-Feb-2312、人乱于心，不宽余请。2023/2/32023/2/32023/2/3Friday,February3,202313、生气是拿别人做错的事来惩罚自己。2023/2/32023/2/32023/2/32023/2/32/3/202314、抱最大的希望，作最大的努力。03二月20232023/2/32023/2/32023/2/315、一个人炫耀什么，说明他内心缺少什么。。二月232023/2/32023/2/32023/2/32/3/202316、业余生活要有意义，不要越轨。2023/2/32023/2/303February202317、一个人即使已登上顶峰，也仍要自强不息。2023/2/32023/2/32023/2/32023/2/3AimoftheresearchAmethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.AnalysisFlowcytometry(FCM)Confocallaserscanningmicroscope(CLSM)Isolation

Fluorescence-activatedcellsorting(FACS)12129、人的价值，在招收诱惑的一瞬间被决定。2023/2/32023/2/3Friday,February3,202310、低头要有勇气，抬头要有低气。2023/2/32023/2/32023/2/32/3/20234:58:04PM11、人总是珍惜为得到。2023/2/32023/2/32023/2/3Feb-2303-Feb-2312、人乱于心，不宽余请。2023/2/32023/2/32023/2/3Friday,February3,202313、生气是拿别人做错的事来惩罚自己。2023/2/32023/2/32023/2/32023/2/32/3/202314、抱最大的希望，作最大的努力。03二月20232023/2/32023/2/32023/2/315、一个人炫耀什么，说明他内心缺少什么。。二月232023/2/32023/2/32023/2/32/3/202316、业余生活要有意义，不要越轨。2023/2/32023/2/303February202317、一个人即使已登上顶峰，也仍要自强不息。2023/2/32023/2/32023/2/32023/2/3AimoftheresearchAnalysisFlowcytometry(FCM)Confocallaserscanningmicroscope(CLSM)BeckmanCoulter--MoFlo™XDPAnalysisrate:100,000events/sec.Sortrate:70,000events/sec.14Amethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.14AimoftheresearchAnalysisFlowcytometry(FCM)Confocallaserscanningmicroscope(CLSM)QuantitiveHighresolutionimaging15Amethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.15AimoftheresearchAnalysisFlowcytometry(FCM)Confocallaserscanningmicroscope(CLSM)ImagingFlowCytometer(Amnis)12ImagesforeverycellQuantitiveHighresolutionimaging16Amethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.16AimoftheresearchIsolation

Fluorescence-activatedcellsorting(FACS)Apassingcellcanbeselectivelyadddifferentcharge,fallingintodifferentcontainers.17Amethodforanalysisandisolationfree-floatingadipocytesbyFlowCytometry.17DifficultyAdipocytesarelargeandfragile50–200μmEasytobebroken,whentheflowrate(pressure)islarge.1818DifficultyAdipocytesarelargeandfragile50–200μmEasytobebroken,whentheflowrate(pressure)islarge.ContaminantsStromal/vascularcellsAggregatesofadipocytesFreelipiddropletsOtherdebris1919SolutionAdipocytesarelargeandfragile50–200μm——usingflowcytometrywithlargerinternaldiameterofthefluidics(150to250μm).Easytobebroken——controlFlowpressuresto5~10psi,slowbutnotbroken.Goodforisolation.2020SolutionContaminantsStromal/vascularcellsAggregatesofadipocytesFreelipiddropletsOtherdebris

——ExclusionofContaminants(4steps)2121SolutionCollagenase——DigestionFiltration——Undigestedtissuefragmentsareexcluded.Centrifugation——Separatedfromstromal/vascularcellsFragile——UserockingratherthanvortexingPreparationofsinglecellsuspensionoffree-floatingadipocytes2222SolutionStep1.Initialseparation——bysizeAdipocytes(50–200μm)biggerthanstromal/vascularcells(<20μm)—higherinForwardScatter(FSC)Thepresenceofhighlyrefractilelipiddropletsinadipocyte—higherinSSCOverlap——needfurtherseparationSmalladipocytes——ignored

(entireanalyzeifnecessary)2323SolutionContaminantsStromal/vascularcellsAggregatesofadipocytesFreelipiddropletsOtherdebris——ExclusionofContaminants(4steps)2424SolutionStep2.ExclusionofaggregatesandselectionofsingletsAggregatescanbeidentifiedbydotplot(WidthorArea).3parameters:

Height

—voltage,Width—passingtime(size),Area=HxWWhenaggregates(doublets)pass,spendmoretime,Wincreased2525SolutionStep2.ExclusionofaggregatesandselectionofsingletsStainnucleibyDyeCycleViolet(DCV)fluorescence.

Singlecellscanbeeasilygatedfromaggregates&otherdebrisThisstepcouldn’texcluderemainingstromal/vascularcells.2626SolutionStep3.Exclusionofsinglestromal/vascularcellsStainthelipiddropletsbyLipidTOXDeepRed(LpdTX)

PositiveSelectionofeventscontaininglipiddroplets2727SolutionStep4:ExclusionofremainingstromalcontaminantsAsmallprobabilityevent:Somestromalcellsmayadheretothefreelipiddroplets.Theremainingstromalcontaminantsbearingthefluorescentantibodies(phycoerythrin(PE)-conjugated)tostromallineagemarkersarestainedtoexclude.(Negativeselection)Negativeselection2828SolutionValidationqRT-PCRStromalcellsmarker(suchasCD34)Black--beforeflowsorting(Highexpressed)White--afterflowsorting(Almostundetectable)2929AnalysisandIsolationofAdipocytesbyFlowCytometryYangLI309、人的价值，在招收诱惑的一瞬间被决定。03-2月-2303-2月-23Friday,February3,202310、低头要有勇气，抬头要有低气。***2/3/20234:58:04PM11、人总是珍惜为得到。03-2月-23**Feb-2303-Feb-2312、人乱于心，不宽余请。***Friday,February3,202313、生气是拿别人做错的事来惩罚自己。03-2月-2303-2月-23**03February202314