| BackgroundInformation | ApplicationCHEMICON"sNS225NeuriteOutgrowthAssayKit(1μm)utilizesmicroporoustissuecultureinserttechnologyfromMilliporeandisbasedontheuseofMillicellcellcultureinserts(chambers)containingapermeablemembranewith1μmporesatthebase.Theinsertsaredesignedtofitintoareceivervessel,whichwheninusecontainsdifferentiationmediaincontactwiththebottomoftheinsertmembrane.Thepermeablemembranesallowfordiscriminationbetweenneuritesandcellbodies,asprojectingneuriteswillpasseasilythroughtheporesbutcellbodieswillnot.Therefore,byinducingneuritestotraversethesemembranepores,apurifiedpopulationofneuritesislocatedontheundersideofthemembrane,distaltothesurfaceonwhichneuralcellbodiesaredeposited.ThiskithashasbeenspecificallydesignedforandvalidatedwithPC12cells.ThediameterofPC12cellbodiesisrelativelysmallcomparedtothatofmanyothercelltypescommonlyusedinneuriteoutgrowthassays[5,6,7].Asaresult,manyPC12cellbodiesmaypassthoughporeslargerthan1μm,significantlyobscuringneuriteoutgrowthassayresults.Usinga1μmporesizegreatlyreducesbackgroundandimprovesthesignal-to-noiseratioinstudiesusingPC12cells.Useofthis1μmporesizewithlargercelltypesthatextendwiderdiameterneurites,e.g.,N1E-115cells,willgreatlydiminishthenumberofneuritesabletoprojectthroughtotheundersideofthemembrane,andisthusnotrecommended.Theassayisasimple,efficient,andversatilesystemforthequantitativedeterminationofcompoundsthatinfluenceneuriteformationandrepulsion.Withthissystem,itispossIBLetoscreennumerousBIOLOGicalandpharmacologicalagentssimultaneously,directlyevaluateadhesionandguidancereceptorfunctionsresponsibleforneuriteextensionandrepulsion,aswellasanalyzegenefunctionintransfectedcells.Themicroporousfilterallowsforbiochemicalseparationandpurificationofneuritesandcellbodiesfordetailedmolecularanalysisofproteinexpression,signaltransductionprocessesandidentificationofdrugtargetsthatregulateneuriteoutgrowthorretractionprocesses.ReagentsandmaterialssuppliedintheNS225NeuriteOutgrowthAssayKitaresufficientfor12tests. |
| 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) |
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