Background

Cell cycle analysis by flow cytometry is mainly based on measurement of DNA content by stain with propidium iodide (PI). The stoichiometric nature of PI ensures the accurate quantification of DNA content and allows us to reveal the distribution of cells in G₁, S and G₂ cell cycle stage or in sub-G₁ cell death stage, the latter of which is characterized by DNA fragmentation. Most of the commonly used methods for PI-based DNA quantification require fixation using alcohol or aldehyde, which is time-consuming and labor-intensive. In addition, these methods are incompatible with staining of mitotic markers by fluorescent-labeled antibodies. Therefore, we adopted and optimized a previous established hypotonic buffer to permeabilize cells, allowing simultaneous staining of DNA with PI and mitotic marker, phospho-Histone H3 (pH3), with pH3 specific antibody (Riccardi et al., 2006; Liu et al., 2016). This method enables a rapid (within 20 min) and comprehensive analysis of cell cycle profile (G₁, S, G₂ and M phase).