Attached are three methods for flow cytometric assessment of cell cycle using propdium iodide (PI) and 4′, 6-diamidino-2-phenylindole (DAPI), the most commonly utilized DNA dyes. The PI methods utilize two different preparation techniques; one uses whole fixed cells, the other employs a hypotonic lysing solution to obtain cell nuclei. (See “Troubleshooting” below to determine best option for your test system).
There are numerous additional dyes available for performing cell cycle analysis; for a complete discussion of the dyes, their nucleic acid binding characteristics, excitation/emission wavelengths, and possible uses, see sections 4 and 7 in Current Protocols in Cytometry, eds. J. Paul Robinson et al, Wiley.
One parameter DNA profiles are a relatively easy, quick and cheap assessment of cellular proliferation. However data interpretation can be difficult, especially in drug treated samples where the mode of action of the drug is some type of cell cycle arrest. In many situations it is strongly recommended to add other stains like cyclins or pH3 to the analysis for more specific interpretation of the different phases of cell cycle progression
Technical Considerations:
- Data evaluation: Assess data obtained using the following criteria. If not satisfied with results, consider the troubleshooting tips below for determining a possible root cause:
- Coefficient of Variation (CV) of G0/G1 population: In principle, since all cells in G0/G1 have the same DNA content, the dye should bind equally and variation in the fluorescence should be minimal. Realistically, there are some imperfections in detection and preparation techniques which cause variation in fluorescence detection. Assuming instrument alignment is satisfactory, a guideline would be to aim for a CV of 4% for cell lines and 2% for primary cells.
- G2:G1 ratio: This should be close to 2.0 on a linear scale for a given cell cycle result. Lower numbers may suggest incomplete saturation of the chosen dye.
- Low percentage of cell aggregates: As assessed by software evaluation or hardware exclusion, aggregates should pose minimal interference with sample analysis.
- Minimal debris: In non-apoptotic fresh cell populations, there should be a minimum of unstained events.
- Cell number: Typically, one should collect at minimum 30,000 cells per cell cycle distribution for accurate assessment of the cell cycle fractions.
- Data Interpretation: Accurate assessment of the number of cells in each cell cycle fraction is made by analyzing the data using software which employs a data-fitting algorithm. Our lab uses Modfit LT (Verity Software House, Topsham, ME) for this purpose.
Troubleshooting:
- Cell suspension: Cells must be in a single cell suspension prior to any fixation and staining for cell cycle analysis as aggregates will impact your data interpretation. While both the cytometric and data analysis procedures to evaluate results have mechanisms to exclude aggregates, no method for eliminating them is perfect. Especially if using cells from a monolayer, be sure to evaluate that cells are completely disaggregated prior to processing.
- Cell number: An excess of cells will interfere with saturation of the DNA staining and result in wide CVs; too few will interfere with data interpretation.
- RNase: When utilizing a nucleic acid dye such as propidium iodide which is not specific for DNA, it is essential to use a DNAse free RNAse preparation according to recommended concentration in procedure.
- Dye concentration: Although most cell cycle procedures come with a recommended concentration of dye, it is useful to titer the dye concentration for your cell type to attain best dye saturation (see “Technical Considerations”).
- Whole cell vs. Nuclear cell preparation: If above troubleshooting categories do not improve your data, consider utilizing a nuclear cell preparation –see protocols below.