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1、SUPPLEMENTARYINFORMATIONImmunocytochemistryAfterfixationin4%paraformaldehyde,immunostainingforBMPR-1A,-1Band-2wascarriedoutaccordingtothemanufacturer’sinstructions(1:50R&DSystems).Whenstainedforphospho-Smad1(1:100CellSignaling,Beverly,MA),cellsweretreatedwithBMP4(100ng/ml)atdifferenttimes
2、(from5minutesto2hours).Appropriateisotypicornegativecontrolswerealwaysincludedthroughouttheseprocedures.Apoptoticcellsweredetectedusingdigoxigenin-basedmodificationoftheoriginalTUNELmethodintroducedby1usingthefluorescein-dUTPTUNELassay(InSituCellDeathDetectionKit,Fluorescein,RocheAppliedS
3、cience).Briefly,cellsgrownon12-mmcoverslipswerefixedin4%paraformaldehydefor10minatroomtemperatureandthenrinsedinPBS.Cellswerethenpermeabilizedfor2minonicebeforelabellingwith50mlofTUNELreactionmixtureandincubatingat37°Cfor1hourinahumidifiedchamberunderparafilmcoverslips.AfterwashingwithPBS
4、,slidesweremountedinDAPI-containingVectashieldTMandexaminedbyfluorescencemicroscopy.Forpropidiumiodide(PI)staining,cellswerefixedin4%paraformaldehydefor10minatroomtemperature,rinsedinPBSandincubatedwithPI(1mg/ml)for5minatroomtemperature.Afterwashing,coverslipsweremountedinDAPI-containingV
5、ectashieldTMandanalysedbyfluorescencemicroscopy.PIexclusiondenotedviablecells.Foralloftheaboveassays,sampleswereruninsixreplicatesforeachconditiontested.Conventionalandreal-timePCRTotalRNAwasisolatedfromculturedandacutelydissociatedGBMcellsusingTRIzolreagent(LifeTechnologies,Rockville,MD)
6、,andreverse-transcribedusingSuperScriptRNAseH-ReverseTranscriptase(LifeTechnologies).TheamountsofcDNAusedastemplatesintheconventionalPCRreactionswerenormalizedwithreferencetoglyceraldehyde-3-phosphatedehydrogenase(GAPDH).MCF-7celllineswereusedaspositivecontrolsforBMPRs.PCRproductswerevisu
7、alizedbyelectrophoresisinagarose(1%)gelsstainedwithethidiumbromide.QuantitativeRT-PCRreactionswererunintriplicateusingBrilliant®SYBR®GreenQPCRCoreReagentKit(Stratagene,LaJolla,CA).SYBRGreendyebindstoanyPCRproduct,andthereforedoesnotrequiretheuseofsequence-specificpr
