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1、Chapter16High-ThroughputHigh-ContentFunctionalImageAnalysisofNeuronalProteinsImplicatedinParkinson’sDiseaseEvaBlaasandRonaldE.vanKesterenAbstractParkinson’sdisease(PD)ischaracterizedbytheprogressivelossofdopamineneurons.Here,wedescribehowtousehumanSH-SY5Yneuroblastomacell
2、sasaninvitrocellmodeltostudytheeffectsofcandi-datePDsusceptibilitygenesondopamineneurondifferentiationandviability.ThiscellmodelcanbeusedtostudytheeffectsofsiRNA-inducedgeneknockdown,eitheraloneorincombinationwithPD-relatedcellularstressorssuchasMPP+,andiscompatiblewithhi
3、gh-throughputcellularscreeningplatformssuchastheCellomicsArrayScanVTIHCSReader.Keywords:Parkinson’sdisease,Neurodegeneration,Cellularscreening,High-contentanalysis,SH-SY5Y,1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine1.IntroductionRecentgenomicsandproteomicsstudiesimplicat
4、emanygeneproductsasthetargetmoleculesfortheexpression/maintenanceofvariousneurodegenerativeorpsychiatricdisorders.Thesedis-ordersarecomplexandinvolvemanycellularmechanisms.Thereisaneedforhigh-throughputhigh-contentassaystosystemati-callyassessthefunctionalcontributionofth
5、esegenestoknownpathogenicmechanisms.Automaticimagingsystemshavebeendevelopedforthispurposeandappliedfortheanalysisofmanycelltypes.TheThermoScientificCellomics®ArrayScan®VTIisamodularhigh-contentscreeninginstrumentdesignedforhigh-throughputfluorescenceimagingandquantitativ
6、eanalysisoffixedandlivecells.Cellcultureson,amongothers,a96-wellformatcanbeanalyzedautomatically.Anaverageofsingle-cellmorphologyisKaWanLi(ed.),Neuroproteomics,Neuromethods,vol.57,DOI10.1007/978-1-61779-111-6_16,©SpringerScience+BusinessMedia,LLC2011211212BlaasandvanKeste
7、renobtainedbyscanningthecellsatdifferentlocationsofeachwell,whereeachwellissystematicallydividedintofields.Automaticfocussingoftheimagesisacquiredbytheirradiationofalaserfromasourcenexttothemicroscopelenstothebottomofthe96-wellplate.Uptofivedifferentfilterscanbeusedtodiff
8、erenti-atefivedifferentcellularcomponentswithinonecell.Werou-tinelyuseXF93-Hoechst,XF93-TRITC,XF
