Cell Cycle Analysis Calculator
Calculate G1, S, G2/M fractions from flow cytometry DNA content data.
How Flow Cytometry Reads the Cell Cycle
Propidium iodide binds DNA stoichiometrically, so fluorescence intensity is proportional to DNA content. That gives a characteristic histogram with two peaks and a valley between them.
| Population | DNA content | Position |
|---|---|---|
| Sub-G1 | < 2n | Left of the G1 peak — apoptotic |
| G0/G1 | 2n | First peak |
| S phase | Between 2n and 4n | Valley between peaks |
| G2/M | 4n | Second peak, twice the G1 intensity |
The sub-G1 population is diagnostically useful: apoptotic cells have fragmented their DNA, so some is lost during permeabilisation and they stain below 2n. A growing sub-G1 fraction is a standard apoptosis readout.
What DNA Content Cannot Distinguish
Two important limitations follow from measuring DNA alone.
| Cannot separate | Why | Solution |
|---|---|---|
| G0 from G1 | Both are 2n | Ki-67 staining marks cycling cells |
| G2 from M | Both are 4n | Phospho-histone H3 marks mitosis |
| Early S from late G1 | Overlapping intensity | BrdU or EdU incorporation |
This matters when interpreting arrest. A drug that increases the 4n population may be arresting cells in G2 or in mitosis, and those are different mechanisms — taxol blocks in mitosis while a DNA damage checkpoint blocks in G2. Phospho-histone H3 separates them.
Interpreting Arrest Patterns
| Pattern | Suggests |
|---|---|
| G1 accumulation | p53 activation, CDK4/6 inhibition, contact inhibition |
| S phase accumulation | Replication stress, antimetabolites |
| G2/M accumulation | DNA damage checkpoint, spindle poisons |
| Rising sub-G1 | Apoptosis |
EdU has largely replaced BrdU for S-phase labelling. BrdU detection requires acid or heat denaturation to expose the epitope, which damages other antigens and the DNA stain. EdU is detected by click chemistry with no denaturation, so multiplexing works far better.
Worked Examples
Common Mistakes
DNA content cannot distinguish G2 from M. Phospho-histone H3 staining is needed to identify genuinely mitotic cells.
Both are 2n. Quiescent and cycling cells look identical unless a proliferation marker such as Ki-67 is included.
It reflects apoptotic cells with fragmented DNA and is a useful readout that is easy to gate out accidentally.
BrdU detection needs harsh denaturation that damages other epitopes and DNA staining. EdU click chemistry avoids this entirely.