Hi,

There could be plenty of reasons for your cells to grow slower. It depends on the cell type, compound you use as a treatment, growth media, etc. The broadest way to get an idea about what is going on is to perform RNA-Seq and ATAC-Seq to get a transcriptome and chromatin accessibility. Differentially expressed genes and/or accessible regions will give you a hint about the reason for decreased proliferation.

Anyway, if you want to proof the slowed cell cycle, you might use something like FUCCI live-cell staining while recording the growth with IncuCyte in all necessary channels.

Best,

Michail

Thanks for your answer. Indeed, I will perform RNAseq and I'm not looking for the exact reason of the slower proliferation but rather for thinks like slowed cell cylce/ cell cylce arrest...(Maybe my question was a bit unprecise in that matter)

So the method you suggested is probably one to answer my question. The only problem: My cells already express GFP, which would interfere with the FUCCI right?

It probably will. I am not sure if there are FUCCI kits available in other colours (it is Red/Green). Anyway, it also depends on the localisation of your GFP (FUCCI is nuclear) and relative intensity of the signals. In any cases, even if you have green channel blocked by GFP, you may still see decreased appearance of the red signal, which also will proof slowed cell cycle, just not so elegant, as with both colours...

Best,

Michail

May be it is possible to make analysis of cell cycle with flow cytometry using BrdU (5'-bromo-2'-deoxyuridine)?I know that the time-lapse measurement of the cohort of BrdU-labeled cells allows estimating their rate of progression through different points of the cell cycle. May be it could proof slower cell cycle.

Terry, N. H. A. and White, R. A. (2001) Cell cycle kinetics estimated by analysis of bromodeoxyuridine incorporation. Meth. Cell Biol. 63, 355–374.

Have you looked at markers for ferroptosis?. this is independent of apoptosis pathway.