Cell Cycle Synchronization and Flow Cytometry Analysis of Mammalian Cells
Analysis of cellular DNA content and measurement of pulse-labeled newly replicated DNA by flow cytometry are useful techniques for cell cycle studies. In this chapter, we describe the protocols for cell cycle synchronization of mammalian cells, including time course designs and consideration of cell types to achieve successful experiments, along with the methods for detection of DNA. Some selected applications dealing with siRNA-mediated knockdown are also presented.
Key wordsCell cycle synchronization Flow cytometry Double-thymidine block Mitosis DNA replication Cell cycle inhibitors BrdU incorporation Click chemistry Embryonic stem cells siRNA-mediated gene knockdown
This work was supported in part by The Naito Foundation Continuation Subsidy for Outstanding Projects (H.M.) and JSPS KAKENHI grant 23580243 (N.Y.-S.).
- 2.Juan G, Darzynkiewicz Z (1998) Cell cycle analysis by flow and scanning cytometry. In: Celis JE, Hunter T, Carter N, Shotton D, Small JV, Simons K (eds) Cell biology: a laboratory handbook. Cell and tissue culture and associated techniques, 2nd edn. Academic Press, San Diego, pp 261–274Google Scholar
- 5.Davies D, Allen P (2007) DNA analysis by flow cytometry. In: Macey MG (ed) Flow cytometry: principles and applications, 2007 edn. Humana Press, New York, pp 165–180Google Scholar
- 10.Perea-Martinez I, Nagai T, Chaudhari N (2013) Functional cell types in taste buds have distinct longevities. PLoS One 8(1):e53399Google Scholar
- 11.Bendall SC, Simonds EF, Qiu P, el Amir AD, Krutzik PO, Finck R, Bruggner RV, Melamed R, Trejo A, Ornatsky OI, Balderas RS, Plevritis SK, Sachs K, Pe’er D, Tanner SD, Nolan GP (2011) Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum. Science 332(6030):687–696PubMedCentralPubMedCrossRefGoogle Scholar