腫瘤干細(xì)胞的形成非粘連球體能力分析
Sphere-forming assays.Cultures were trypsinized and strained through a 40-μm sieve.Tumors were minced,digested in 4 mg/mL collagenase 3 (Worthington,Freehold,NJ),2 mg/mL hyaluronidase (Sigma-Aldrich,Oakville,Ontario,Canada),and 2 mg/mL collagenase IV (Sigma) in serum-free DMEM for 25 min at 37°C,strained through a 40-μm sieve,incubated 5 min at room temperature in 0.75% NH4Cl to lyse RBC,and washed with DMEM + 10% FBS.Single cell suspensions were confirmed microscopically,washed with PBS,and suspended at 1,000 cells/mL in serum-free DMEM supplemented as described ( 4).One hundred microliters of the cell suspension was plated in each of the 30 wells of a 96-well microplate (Nunc).Each well was fed 25 μL serum-free supplemented DMEM every other day for 11 days (from culture) or 14 days (from tumor).Spheres (tight,spherical,nonadherent masses >90 μm in diameter) per well were counted,and at least 50 spheres per group were measured with an ocular micrometer.For secondary sphere-forming assays,primary spheres were dissociated mechanically and processed as in the primary assay.