To overcome this limitation, some alternative methods have been developed. *cell permeable dye which can stain DNA in both live or fixed cells. However, this method is insufficient to understand detailed cell cycle status because DNA content alone cannot distinguish resting/quiescent cells (G0) from G1 phase cells. Since then, many fluorescent DNA dyes have been developed for multiplex analysis of cellular DNA content and other proliferation-related markers ( Table 1). In 1969, the feulgen-DNA staining method was first described to analyze cell cycle distribution ( Van Dilla et al., 1969). This reveals a snapshot of cell cycle status among 3 distinct groups ( i.e., G0/G1 (2n), S (2n~4n), and G2/M (4n) phase, respectively). The earliest and simplest approach to analyze cell cycle status is to measure cellular DNA content at a single time point ( Darzynkiewicz and Huang, 2004). This resting state is also referred as quiescent ( Zetterberg et al., 1995). These resting cells are characterized by having minimal cell cycle machinery and maintaining specialized cellular functions rather than by proceeding to cell proliferation. However, under certain circumstances, cells can enter G0 phase, where the cells are neither dividing nor preparing for proliferation. During cell cycle progression, proliferating cells sequentially undergo a transition of G1→S→G2→M phases for synthesis of DNA, preparation of cell division and subsequent mitosis process ( Malumbres and Barbacid, 2009). This enables one to investigate underlying basic mechanisms as well as to evaluate therapeutic efficacies of anti-cancer drugs.

Assessing cell cycle distribution and cell proliferation is important for studying cell growth differentiation, senescence and apoptosis.

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