DescriptionCancercellsdonotshowanchorageandcontactinhibitionofgrowth.Toassesstheanchorageandcontactindependentgrowthofcells,nobleagarassaycanbeutilized.Nobleagarisusedtocoatthetissuecultureplatestocoverthepolylysinecoatingoftheplate.Onthisnobleagarcoated,anchorageindependenttissuecultureplatesnormalandcancercellswillbeseededinverylowdensitytopreventthecontactbetweencells.PlatingefficiencyisameasureoftheclonogenicABIlityofcellsandCancercellswillhavehigherplatingefficienciesthannormalcells.Procedure1.Make1.2%and0.6%nobleagarmediumindw.2.Autoclavebothnobleagarmediumamdequilibrateat42degreecelsius.3.Prepare20%FBScontainingDMEM/RIPAoranyothertissueculturemediumaccordingtoyourcells.Thesetissueculturemediumshouldnotcontainphenolred.4.Alsoequilibratethetissueculturemediumat42degreecelsius.5.Mixthe1.2%nobleagarand20%DMEMmediumandprecoatthe6mmpolystyreneflatbottomoftissuecultureplateswith0.6%nobleagarinDMEMsupplementedwith10%FBS.6.Allowittosolidifyfor5-7min.7.Mixthe0.6%nobleagarand20%DMEMmediumtomake0.3%nobleagarinDMEMsupplementedwith10%FBS.8.HarvestcellsfromtissuecultureflasksbyTrypsin/EDTA.9.WashthecellswithPBS.10.ResUSPendthecellsin0.3%nobleagarinDMEMmediumcontaining10%FBSatadensityof1500cells/ml.11.Plate4500cellson0.6%coatedtissuecultureplatesbypouringthe3mlofcellsin0.3%nobleagarinDMEMmediumcontaining10%FBS.12.Allowthistosolidifyfor5-7min.andthencoveritwith20%FBScontainingDMEMtissueculturemedium.13.Incubatetheplatesat37degreecelsiusfor2-3weeks.14.Changethe20%FBScontainingDMEMmediumtwiceaweek.15.Numberofcloneswillbecountedbyphasecontrastmicroscopyafter14-21days.

================  蚂蚁淘在线  ================

免责声明：本文仅代表作者个人观点，与本网无关。其创作性以及文中陈述文字和内容未经本站证实，对本文以及其中全部或者部分内容、文字的真实性、完整性、及时性本站不做任何保证或承诺，请读者仅作参考，并请自行核实相关内容

版权声明：未经蚂蚁淘在线授权不得转载、摘编或利用其他方式使用上述作品。已经经本网授权使用作品的，应该授权范围内使用，并注明“来源：蚂蚁淘在线”。违反上述声明者，本网将追究其相关法律责任。