Cell Cycle Analysis - Propidium Iodide
Although propidium iodide is probably the most commonly used dye to quantitatively assess DNA content, there are several different dyes that are available which bind stoichiometrically to DNA. These may be divided as follows:- UV excited: Hoechst 33342, Hoechst 33258, DAPI
- 457nm excited: Mithramycin
- 488nm excited: Propidium Iodide, 7Aminoactinomycin D, SYTOX Green, DRAQ5
- 633nm excited: TO-PRO-3 Iodide
One important aspect of DNA analysis is the ability of the cytometer to exclude cell doublets. In a cytometer where the beam diameter is larger than the nuclear size, we can look at pulse width versus pulse area to exclude cell doublets - a true G2 cell will have a smaller width than two G1 cells passing through the beam together consecutively.
Once we have identified the single cell population, we can estimate the percentage of cells in G1, S phase and G2/M either by subjectively applied markers (below, left) or by using a program that will mathematically deconvolute the DNA histogram and thus give a more accurate measurement of the percentage of cells in each phase (below, right using FlowJo).
Sometimes, especially in cells in culture and in cells that have been arrested in G2 we see functional endoreduplication, i.e., cells double their DNA but do not divide leading to a population of 8n cells. Using pulse processing these can be identified and quantitated, although these are often overlooked. In the example below, gating on single cells allows a true octoploid population to be seen - and we can also show that these are cycling by using bromodeoxyuridine.
Protocols for DNA staining are available for:
- Propidium iodide in pdf format
- Mithramicin in pdf format
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