I'm using Abcam 112116 cell cycle assay kit - Green Fluorometric to detect cell cycle for HCT116 and Caco2 (colorectal cell lines). I've performed an optimization run using the recommended protocol (Nuclear green 1:200), and I even included several nuclear green concentrations (1:400, and 1:600).
Cell scattering shows two populations for HCT116 (probably cell duplicates) and minimal separate scattering with Caco2 (for 1:200 only as recommended by the kit). Nevertheless, when I select a single cell population I can't seem to see a normal cell cycle, always one prominent peak is showing.
What could cause the disappearance of cell cycle?
Note the following:
- Untreated unstained samples were used to detect auto-florescence
- Untreated stained cells were used to detect normal cell cycle
- I've used a cytotoxic treatment below IC50 for 24 hrs duration
- Treated stained cells are being investigated for cell cycle arrest compared to untreated ones
- Floating treated cells were collected in the samples
- Floated untreated cells were NOT collected in the samples
- no starvation was done
- Incubation of cells with nuclear green was 50 minutes
- cells are adherent, so trypsinization was done and following by complete media wash prior to incubation of with nuclear green in fresh complete media
- I've used Doxorubacin as a sample, the graph shows shifting but I can't quantify it without distinct cell cycle phases (attached is a full profile scatter for dox).
Should any of the following suggestions be crucial for better cell cycle presentation:
- Starving the cells prior to treatment?
- Using short duration treatment (1:200; recommended is 1:200)?
- Using prolonged incubation time with the dye (>60 minutes; recommended is 30-60 minutes)?
- Using cell strainer to avoid duplicate cells?
- Floating cells in the treated/untreated samples should/not be collected?
Attached is a full scattering profile for the untreated HCT116 cell line. Note that the second peak is probably representing cell duplicates rather than a population of single cells in the G2 phase, despite using a cell strainer.
Thank you
Hi Husam
It could be that DOX is quenching your Nuclear Green signal. Could you try titrating DOX to see if this is the case? I have seen this reported for Hoechst and DOX for imaging applications.. as the DOX conc. was increased the nuclei disappeared, perhaps by a FRET-like mechanism whereas here it maybe displacement/competition. The authors in that case (Antczak et al.) used DRAQ5 as the nuclear counterstain and it can be used here for unfixed or fixed cell cycle analysis.
However, there are lots of caveats to doing cell cycle analysis - read up information from Derek Davies (The Francis Crick Inst.) - he knows all about this methodology.
Regards and good luck,
Roy
Thank you Roy! I'll read through the recommended references.
As for the Dox, Indeed, Dox do interfere with nuclear green signal, but the issue still does exist in the untreated cells, which should exhibit normal cell cycle.
I've performed another optimization using the same conditions and the same cells, except this time I stained the samples with Propidium iodide (PI) only and cell cycle was smoothly expressed with no effort, even when I repeated the experiment across several other cell lines.
I think the kit is the issue.
Hi Husam,
Have you contacted Abcam's tech support?
One other thought is that the cells might have the capacity to clear the Abcam probe (drug resistance mechanism). Perhaps adding 0.5% formaldehyde would "snap" the pumps involved in this without permeabilising your cells or affecting the expression of other cell surface markers. PI will not suffer from this because of the fix, perm, RNase procedure required to use it.
Regards,
Roy
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