Flow cytometry can be used to precisely quantify the amount of DNA in each cell of a sample. This is particularly powerful in assessing the percentage of cells in each stage of the cell cycle: Go/G1 (2N DNA), S-phase (synthesizing DNA) and G2/Mitosis (4N DNA). The determination of cellular DNA content can be done most simply by fixing and permeabilizing the cells with methanol followed by staining the DNA with an appropriate fluorochrome (PI, 7AAD, LDS751, etc. see protocol: Facilities-flow-cytometry-cell-cycle-1). This is referred to as one-dimensional resolution of DNA quantity as it relies exclusively on PI (or an alternative) to report the DNA content. Note that this can be done in conjunction with cell surface or intracellular marker stains if you wish to analyze a specific subset of cells (such as T-cells from a PBMC preparation). For a more simple determination of total number of proliferating cells, one can stain for Ki-67 which is expressed throughout mitosis, but not in G0 cells.
In any assessment of DNA content, it is crucial to eliminate co-incident events and aggregates from analysis (one 4N cell looks the same as two 2N cells going through at the same time, compromising your data). This can be done with singlet gating and by keeping the event rate to <500/second if possible.
One-dimensional determination of DNA content using propidium iodide (PI). Note the contribution of fragmented DNA from apoptotic cells (far left – red arrow). Also note that there is a “smear” of signal above the G2 peak highlighting the critical nature of gating to remove multi-cell aggregates and co-incident events from analysis.
There are several software packages (ModFit, FlowJo, etc) that will fit curves to best estimate the number of cells in the overlapping Go/G1, S and G2/M curves. This cannot be “eyeballed” as small changes in these numbers can have significant consequences.
Since the boundary between the S-phase “peak” and the other two peaks is very difficult to determine and very sensitive to small changes, there are protocols that are more sensitive to identifying these boundaries using two-dimensional fluorescent data (two reporters). The most common is to stain total DNA while at the same time including BrdU/EdU which incorporates into newly synthesized DNA. Older BrdU protocols are harder and harsher than commercially available EdU Click-It kits (Life Technologies), which are a commonly recommended alternative (see protocol: Facilities-flow-cytometry-cell-cycle-2).
Two dimensional analysis of DNA content. BrdU is incorporated into the newly synthesized DNA, thus separating the S-phase population along the Y-axis (blue) while the G0/G1 population (red) can be separated from the G2/M population (yellow) which has more PI/DAPI/etc. staining because it contains twice the DNA
It is also possibly to use these techniques to investigate aneuploidy by comparing the DNA content of the Go/G1 peak from control and suspected aneuploid cells to see if the DNA content (DNA index) is different (translocations, etc). This is summarized as an addendum to the PI protocol as determination of DNA index.
Please keep in mind that these procedures are merely general suggestions and there are several alternatives to either one-dimensional or two-dimensional resolution of DNA content to suit your cell type, co-staining fluorophores, etc. If the above protocols are not suitable, please talk to the flow cytometry core staff that will be happy to assist.
