Matrigelinvasionassays

PostedonSaturday,October18,2003DescriptionMatirgelisconsideredasbasementmembraneandgeneratedfromEHSsarcoma.Matrigelcontainsnotonlybasementmembranecomponents(collagens,laminin,andproteoglycans)butalsomatrixdegrADIngenzymes/theirinhibitorsandgrowthfactors.InvasionoftumorcellsintoMatrigelhasbeenusedtocharacterizeinvolvementofECMreceptorsandmatrixdegradingenzymeswhichplayrolesintumorprogression.Procedure1.ThawMatrigelat4Covernight.2.DiluteMatrigel(5mg/mlto1mg/ml)inserumfree-coldcellculturemedia(RPMI1640,EMEM,DMEM,etc).3.Put100ulofthedilutedmatrigelintoupperchamberof24-welltranswell4.Incubatethetranswellat37Catleast4to5hforgelling.5.HarvestcellsfromtissuecultureflasksbyTrypsin/EDTA.6.Washthecells3timeswithculturemedia(RPMI1640,EMEM,DMEMetc)containing1%FBS.7.ResUSPendthecellsinmediacontaining1%FBSatadensityof10^6cells/ml.8.Gentlywashgelledmatrigelwithwarmedserumfree-culturemedia.9.Put100ulofthecellsuspensionontothematrigel.10.lowerchamberofthetranswellisfilledwith600ulofculturemediacontaining5ug/mlfibronectin,asanadhesivesubtrate.11.Incubateat37Cfor20to24h.12.Removetranswellsfrom24-wellplatesandstainedwithDiff-Quicksolution.13.Scrapeoffnoninvadedcellsonthetopofthetranswellwithacottonswab.14.Countinvadedcellsunderalightmicroscope.Recipes-Matrigel(Becton-dickinson)-24-transwell(Coster)-Fibronectin(Sigma)-Diff-Quickstainingsolution(FischerScientific)