Overview | Application
CHEMICON"sNS220NeuriteOutgrowthAssayKit(3μm)utilizesmicroporoustissuecultureinserttechnologyfromMilliporeandisbasedontheuseofMillicellcellcultureinserts(chambers)containingapermeablemembranewith3-μmporesatthebase.TheMillicellinsertsutilizedinthiskitareappropriateforcellsprojectingneuritesofupto3μmdiameter,e.g.N1E-115cells[Abeetal.,2003],DorsalRootGangliaandSchwanncells[Windebanketal.,1985].Theinsertsaredesignedtofitintoareceivervessel,whichwheninusecontainsdifferentiationmediaincontactwiththebottomoftheinsertmembrane.Thepermeablemembranesallowfordiscriminationbetweenneuritesandcellbodies,asprojectingneuriteswillpasseasilythroughtheporesbutcellbodieswillnot.Therefore,byinducingneuritestotraversethesemembranepores,apurifiedpopulationofneuritesislocatedontheundersideofthemembrane,distaltothesurfaceonwhichneuralcellbodiesaredeposited. Theassayisasimple,efficient,andversatilesystemforthequantitativedeterminationofcompoundsthatinfluenceneuriteformationandrepulsion.Withthissystem,itispossIBLetoscreennumerousBIOLOGicalandpharmacologicalagentssimultaneously,directlyevaluateadhesionandguidancereceptorfunctionsresponsibleforneuriteextensionandrepulsion,aswellasanalyzegenefunctionintransfectedcells.Themicroporousfilterallowsforbiochemicalseparationandpurificationofneuritesandcellbodiesfordetailedmolecularanalysisofproteinexpression,signaltransductionprocessesandidentificationofdrugtargetsthatregulateneuriteoutgrowthorretractionprocesses.ReagentsandmaterialssuppliedintheNS220NeuriteOutgrowthAssayKitaresufficientfor12tests. Note:the3μmporesizeinsertsinthiskitareunsuitableforusewithPC12cells.DuetotherelativelysmalldiameterofPC12cellbodies[Abeetal.,2003],someofthesecellsmaypassthough3μmporesandobscuretheresults.Chemicon"sNS225NeuriteOutgrowthAssayKit(1μm)hasbeenspecificallydesignedforandvalidatedwithPC12cells. |
MaterialsRequiredbutNotDelivered | 1.Desiredextracellularmatrixmembranecoatingprotein,e.g.,bovinecollagenIormouselaminin. 2.Tissuecultureinstruments/supplies(including37°Cincubator,growthmedia,flasks/plates,detachmentbuffer,etc.)forcelltypeofinterest. 3.Differentiationmedia/factorsforcelltypeofinterest(e.g.,nervegrowthfactor,serum-freemedia). 4.1Xphosphatebufferedsaline(PBS)withCa2+/Mg2+. 5.Invertedmicroscopeforneuritevisualizationonmembraneinserts. 6.Parafilmandspectrophotometerforneuritestainextractionandquantification. 7.Proteinlysisbuffer(optional) |