Hi folks!
I've been struggling a bit with understanding cell cycle assays, i.e. EdU assays. We do them in our lab with the click-it kit from thermo, using EdU and FxCycle (stains total dsDNA) but there's a basic feature of those assays I don't understand.
EdU as I understand it, is incorporated into newly synthesized DNA and therefore the brightness of the fluorophore used to mark EdU is proportional to the amount of newly synthesized DNA during the pulse.
FxCycle should label all dsDNA so an increase in EdU should always be accompanied with an increase in FxCycle but that's not the case! In what I assume should be early S phase, the cells first increase in EdU and only when they've reached almost maximum MFI in the 'EdU channel' they start increasing in FxCycle. That's something we can see in our own plots as well as in resources available on the net and papers (e.g. https://www.bdbiosciences.com/documents/BD_FACSVerse_CellCycleAnalysis_AppNote.pdf)
Can someone help me understand this? How/why would a cell massively synthesized DNA without (apparently) increasing the amount of total dsDNA? What would it mean if the cells get stuck at this stage (very brigth EdU but still FxCycle dim)? Is there a technical aspect of the FxCycle labelling that I'm maybe missing?
Any help is greatly appreciated!
It's because the proportion of the amount of DNA synthesised during the EdU labelling period is small compared with the total (=genomic) DNA.
Don't be deceived by the scales of your plot. If you could plot "absolute amount of DNA", the incorporated EdU is minute compared with genomic DNA.
Shin-Ichiro Hiraga That's a great point, thank you a lot for your help! However, it doesn't really explain the reverse phenomenon; why one sees an increase in *only* FxCycle but not EdU during the course of the S-phase. How can the total mount of genomic DNA increase without any DNA being synthesized?
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