Cell cycle refers to the process in which a cell divides and duplicates (see figure at right). With a few exceptions, each cell in an organism contains the same amount of DNA and the same number of chromosomes. Thus, cells must duplicate their content of DNA prior to cell division so that each daughter cell will receive the same amount of DNA as the parental cell. Analysis of cell cycle can be performed by flow cytometry, using a fluorescence dye that will stain DNA. The fluorescence intensity will correlate with the amount of DNA contained in the cells.
Obtaining good results in DNA analysis depends on proper sample preparation and instrument optimization of the optics and fluidics. The resolution of a flow cytometer can be assessed by measuring the coefficient of variation (CV) of a reference particle: the lower the CV, the better the resolution.
Linearity is critical for DNA experiments. To verify the linearity of DNA data, the pulse-area signal is used to measure the amount of DNA fluorescence detected from cells or nuclei. On an instrument that records data linearly, the doublet peak should be located at twice the mean channel of the singlet peak (see figure at left).
Doublet discrimination, or the ability to resolve singlets from aggregates, is also important for DNA experiments. Aggregated cells or nuclei are detected as events that have two or more times the amount of singlet fluorescence. For cell-cycle analysis, it is important to resolve singlets from aggregates because doublets of G0/G1 cells have the same amount of DNA fluorescence as singlet G2+M cells. Therefore, these doublets accumulate in the same fluorescence area channel as singlet G2+M cells.
DNA Analysis Protocols
Fixed cell cycle:
- 4',6-diamidino-2-phenylidole (DAPI) staining, utilizing detergent (PMID: 18770732): unfixed and fixed cells (see figure)
Live cell cycle:
- 5-Bromo-2'-deoxyuridine (BrdU) incorporation: BrdU, is a pyrimidine analog that is incorporated in place of thymidine in the newly synthesized DNA of proliferating cells. After fixation and DNA denaturation, a conjugated anti-BrdU is added to detect the incroporated BrdU by flow cytometry (see figure). This protocol can be use for in vivo (article) and in vitro (PMID: 18770734) experiments. 5-Ethynyl-2'-deoxyuridine (5EdU) is an alternative to BrdU, which does not require harsh denaturation condition to access DNA.
How to Set Up a Cell Cycle Experiment (Flow Cytometry)?
Analysis of data:
Cell Cycle Tutorial FlowJo (by Tree Star): Two dongles (PC and MAC versions) availables to all FCCS users.
ModFit LT Tutorials, scroll down (by VSH): To be used by FCCS staff only.
Last modified January 15 2016 02:12 PM