Question 15 of 20 Intermediate Interpretation
What does a DNA content histogram tell you, and what can distort it?
Short answer
A DNA dye splits cells into G0 or G1 with 2n DNA, S phase between 2n and 4n, and G2 or M with 4n, and a sub-G1 peak suggests fragmented DNA from apoptosis. Doublets, RNA staining, wide peaks from fast acquisition and debris all distort it.
Why
Dyes such as propidium iodide bind DNA in proportion to its amount, so on a linear scale the G1 peak sits at one value and G2 at about twice that. Cells copying their DNA fill the trough between. The histogram is a snapshot.
It cannot tell resting G0 cells from cycling G1 cells, which Kim and Sederstrom separate with Ki-67 or RNA staining. It cannot tell G2 from mitosis without a mitotic marker. Several artifacts mimic biology.
Two G1 cells stuck together have 4n DNA and look like G2, so area against width gating is essential. Propidium iodide also binds double-stranded RNA, so RNase must be in the stain. Running the sample at a high rate widens the peaks, raising the coefficient of variation and blurring S phase.
The protocol suggests fewer than about 400 events per second. A sub-G1 peak means apoptotic DNA fragmentation, but debris also lands there. For example, an apparent G2 arrest after a drug may shrink to half once doublets are removed.
EdU incorporation then measures S phase directly instead of inferring it from the trough.
A strong answer covers
- G1, S and G2 or M read from DNA amount on a linear scale
- Doublets mimic G2 cells
- RNase is needed with propidium iodide
- Low event rate keeps peaks narrow
- Sub-G1 suggests apoptosis but can include debris
Common mistakes
- Reporting a G2 arrest without a doublet discrimination gate
- Treating the G1 peak as quiescent cells without a G0 marker
- Reading every sub-G1 event as an apoptotic cell
The interviewer might ask next
How would you combine EdU with a DNA dye to measure S phase more directly?