Multiparameter cell cycle analysis.
Cell cycle-related cytometry and analysis is an essential experimental paradigm for the cell biology of yeast, mammalian, and drosophila cells. Methods have not changed much for many years. The most common is DNA content analysis, which has been well-published and reviewed. Next most common is analysis of 5-bromo-2-deoxyuridine (BrdU) incorporation, detected by specific antibodies - also well-published and reviewed. A new measurement approach to S phase labeling utilizes 5'-ethynyl-2'-deoxyuridine (EdU) incorporation and a chemical reaction to label substituted DNA. The approach is new, but published work indicates that it is equivalent to BrdU incorporation. Finally, multiple antibody labeling to detect epitopes on cell cycle-regulated proteins is the most complex of the cytometric cell cycle assays, requiring knowledge of the chemistry of fixation, the biochemistry of antibody-antigen reactions, and spectral compensation. Because all of this knowledge is relatively well presented, methodologically, in many papers and reviews, this chapter presents a bare-bones Methods section for one mammalian cell type and an extended Notes section, focusing on aspects that are problematic or not well described in the literature.
Cytometry and Imaging Microscopy Core, Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH, USA.
James W
JW
Jacobberger
R Michael
RM
Sramkoski
Tammy
T
Stefan
D000906:Antibodies / Q000378:metabolism; D002453:Cell Cycle; D045744:Cell Line, Tumor; D003625:Data Collection; D003626:Data Display; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D013194:Staining and Labeling
D000906:Antibodies; D005456:Fluorescent Dyes