Proliferation & cell cycle
Cells are fixed (usually in cold 70% ethanol) and stained with propidium iodide plus RNase, so that fluorescence reports DNA content alone; the histogram is fitted into G1, S and G2/M fractions.
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PI cell-cycle staining
Cells are fixed (usually in cold 70% ethanol) and stained with propidium iodide plus RNase, so that fluorescence reports DNA content alone; the histogram is fitted into G1, S and G2/M fractions.
How it differs from the standard method
- Add ethanol dropwise while vortexing to avoid clumps.
- RNase is essential: PI also binds double-stranded RNA.
- Acquire on a linear scale, at low flow rate, and gate out doublets on width against area.
- DAPI or Hoechst-type dyes do the same job on violet or UV lasers without RNase.
1Three questions, three kinds of label
"Are these cells proliferating?" hides three different measurements. A DNA stain such as propidium iodide (PI) asks where each cell is in the cycle right now: a cell in G1 has two copies of the genome, a cell in G2 or mitosis has four, and a cell in S phase has something in between.
DNA content
A snapshot of the phase distribution. Fixed or permeabilised cells, dye in excess, RNA removed; read on a linear scale.
DNA synthesis
The analogue is built into new DNA during the pulse and detected afterwards: click chemistry for EdU, an antibody after DNA denaturation for BrdU.
Division history
A covalently bound dye halves at each division, so generations appear as peaks a factor of two apart on a log scale.
2Reading a DNA histogram
With a stoichiometric dye, fluorescence is proportional to DNA. A growing population gives a tall G1 peak, a smaller G2/M peak at twice the intensity, and a plateau of S-phase cells between.
3Pulse labelling and its timing
An EdU pulse labels every cell that spends any part of the pulse in S phase. If the pulse is short compared with S phase, that is close to the fraction in S.
4Counting generations
CFSE diffuses into cells as a non-fluorescent ester, is cleaved by esterases and reacts with intracellular proteins, so it stays put and is diluted only by division. Resolving generations needs a narrow starting peak (uniform labelling in protein-free buffer), enough starting brightness to stay above autofluorescence for six to eight halvings, and an unstimulated control read on the same day, because cells lose part of their label in the first day without dividing.
Key terms
Practise these as flashcards →Phases before replication (2N), during replication, and after replication through mitosis (4N).
Width of the G1 peak divided by its position; the main quality measure of a DNA histogram.
Removing events made of two cells, which have a longer pulse (larger width) than a single cell with the same total signal.
Dye binding in proportion to DNA, which requires dye in excess over binding sites.
Exposing cells to a nucleotide analogue for a defined, short time so that only cells synthesising DNA in that window incorporate it.
Average divisions per original cell, counting non-dividers or only cells that divided at least once.
Events with less DNA than G1: debris and fragmented apoptotic nuclei.
When to use it
- To find which phase a drug arrests cells in (PI or EdU with DNA content).
- To measure the fraction of cells actively replicating DNA, independent of histogram fitting (EdU pulse).
- To follow how many times lymphocytes or other cells have divided after a stimulus, and what fraction responded (CFSE or similar dyes).
Compared with alternatives
Plate readouts of how many cells are left; they cannot tell growth arrest from death on their own.
When a sub-G1 peak or falling counts suggest the cells are dying rather than arresting.
The instrument, compensation and gating principles all of these assays rely on.
Follows individual cells through division directly, with timing per cell, at much lower throughput.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
Cells are fixed (usually in cold 70% ethanol) and stained with propidium iodide plus RNase, so that fluorescence reports DNA content alone; the histogram is fitted into G1, S and G2/M fractions.
- Add ethanol dropwise while vortexing to avoid clumps.
- RNase is essential: PI also binds double-stranded RNA.
- Acquire on a linear scale, at low flow rate, and gate out doublets on width against area.
- DAPI or Hoechst-type dyes do the same job on violet or UV lasers without RNase.