Cell synchronization by inhibitors of DNA replication induces replication stress and DNA damage response: analysis by flow cytometry.

摘要: Cell synchronization is often achieved by inhibition of DNA replication. The cells cultured in the presence such inhibitors as hydroxyurea, aphidicolin, or thymidine become arrested at entrance to S phase and upon release from block they synchronously progress through S, G(2), M. We recently reported that exposure these concentrations commonly used synchronize cell populations led phosphorylation histone H2AX on Ser139 (induction γH2AX) activation ataxia telangiectasia mutated Rad3-related protein kinase (ATR). These findings imply induction replication stress activates damage response signaling pathways caution about interpreting data obtained with use synchronized way representing unperturbed cells. protocol presented this chapter describes methodology assessment H2AX-Ser139, ATM/ATR substrate Ser/Thr SQ/TQ cluster domains well (ATM) treated Phosphorylation proteins detected individual immunocytochemically phospho-specific antibody (Ab) measured flow cytometry. Concurrent measurement cellular content phosphorylated followed multiparameter cytometric analysis allows one correlate extent their cycle phase.