Rapid Profiling Cell Cycle by Flow Cytometry Using Concurrent Staining of DNA and Mitotic Markers
- Bio Protoc. 2017 Aug 20;7(16):e2517. doi: 10.21769/BioProtoc.2517.
- 1. Center for Childhood Cancer & Blood Diseases, The Research Institute at Nationwide Children's Hospital, Ohio State University, Columbus, OH, USA.
- 2. Department of Microbiology and Immunology, Key Laboratory of Developmental Genes and Human Disease, Ministry of Education, Medical School, Southeast University, Nanjing, China.
- 3. University of Pittsburgh School of Medicine, Pittsburgh, USA.
The flow cytometric quantitation of DNA content by DNA-binding fluorochrome, propidium iodide (PI) is the most widely used method for cell cycle analysis. However, the commonly used methods are time-consuming and labor-intensive and are incompatible with staining of mitotic markers by fluorescent-labeled antibodies. Here, we report an optimized simple protocol for rapid and simultaneous analysis of characteristic mitotic phosphorylated proteins and DNA content, permitting the quantification of cells in Mitosis, G1, S and G2 stage in a single assay. The protocol detailed here employs detergent-based hypotonic solution to rapidly permeabilize cells and allows simultaneous staining of DNA with PI and mitotic marker, phospho-Histone H3, with specific antibody within 20 min. The protocol requires only inexpensive and commercial available reagents and also enables a rapid and complete analysis of cell cycle profile.