Characterization of DNA content, cyclin B1 and phosphorylated histone H3 with direct S‐phase using EdU incorporation in multiparameter testing of cell lines with cell cycle blocking agents

• Published in: The FASEB journal 2008-04, Vol.22 (S2), p.362-362
• Main Authors: Bradford, Jolene A, Clarke, Scott T, Hill, Dani, Chen, Yih‐Tai
• Format: Article
• Language: English
• ISSN: 0892-6638; 1530-6860
• DOI: 10.1096/fasebj.22.2_supplement.362

Detailed characterization of cell cycle is critical in basic and applied immunologic and oncologic studies. By combining measurements of DNA content and DNA synthesis, the cell cycle can be resolved into G0+G1, S, and G2+M phases. By including other key players of cell cycle progression, multiparametric analysis of cell cycle by flow cytometry or imaging cytometry can provide further insights into the mechanistic aspects of pharmaceutical agents. We utilized a recently introduced method for determining direct S‐phase synthesis using the incorporation of a nucleoside analog EdU (5‐ethynyl‐2′‐deoxyuridine) coupled with click chemistry. Combining DNA content and direct S‐phase measurement with cyclin B1 (expressed in late‐S, G2, and M phases of the cell cycle) and phospho‐histone H3 (expressed during mitosis), it is possible to identify different subpopulations and assess the effects of the pharmaceutical agents. An amine‐reactive dye is also utilized to eliminate dead cells from the analysis, which may non‐specifically bind the antibodies used, resulting in five‐parameter flow cytometric analysis. Separate testing using a monomeric cyanine dye in combination with a dead cell marker is used to identify early‐apoptotic and late‐apoptotic/necrotic events. In addition to flow cytometry, we show that automated imaging cytometry can be employed in a similar manner to obtain comparable results.