ForResearchUseOnly.Notforuseindiagnosticortherapeuticprocedures.BDBiosciencesAssayMethodsProtocol:EndothelialCellTubeFormationAssayMaterials:•BDMatrigel™matrix,10ml(BDcat.no.354234);recommendedconcentrationofBDMatrigellot:10mg/mlorgreater.•BDFalcon™24-wellflat-bottomstandardtissueculture-treatedplate(BDcat.no.353047).•Endothelialcellculturemedium(e.g.,EGM-2MV,Lonzacat.no.CC-4147).•Fetalbovineserumorappropriategrowthfactorasanangiogenesisstimulator.•Hanks’BalancedSaltSolution(HBSS)(e.g.,LifeTechnologycat.no.14025-092).•Fluorophore(e.g.,BD™CalceinAMFluorescentDye,10x50µg,BDcat.no.354216).•DMSO.•Humidifiedtissuecultureincubator,37°C,5%CO2atmosphere.•EndothelialcellssuchasHMEC-1,HMVEC,orBDHUVEC-2cells(BDcat.no.354151).•Laminarflowtissueculturehood.•Automatedimager,fluorescencemicroscope,tubequantificationsoftware.Procedure:1.0ReconstitutionofBDMatrigelmatrixColorvariationsmayoccurinfrozenorthawedvialsofBDMatrigelmatrix,rangingfromstrawyellowtodarkredduetotheinteractionofcarbondioxidewiththebicarbonatebufferandphenolred.Variationincolorisnormal,doesnotaffectproductefficacy,andwilldisappearuponequilibrationwith5%CO2.1.1ThawBDMatrigelmatrixovernightoniceat4°C.1.2Oncethawed,swirlvialtoensurethatmaterialisevenlydispersed.1.3Spraytopofvialwith70%ethanolandairdry.1.4Keepproductoniceandhandleusingsteriletechnique.1.5Dispensematerialintoappropriatealiquots,usingpre-cooledpipets,tips,andtubes,andrefreezeimmediately.Avoidmultiplefreezethaws.PrecautionBDMatrigelmatrixwillgelrapidlyat22°Cto35°C.Thawovernightat4°Conice.Keepproductonicebeforeuse,andusepre-cooledpipets,tips,andtubeswhenpreparingBDMatrigelmatrixforuse.Angiogenesisischaracterizedbyanumberofcellulareventsincludingendothelialcellmigration,invasionanddifferentiationintocapillaries.Invitroendothelialtubeformationassaysareusedasamodelforstudyingendothelialdifferentiationandmodulationofendothelialtubeformationbyantiangiogenicagents.Imageacquisitionandquantificationoffluorescentlylabeledcellscanbeachieve...
