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Additional peaks in cell cycle assay - Can 37C incubation cause clumping of fixed cells? (Mar/21/2011 )

Hi!

I am perfoming cell cycle measurements with primary cardiac fibroblasts. Recently, I have added an RNase treatment step (37C, 30min) to my protocol, which now gives me much sharper peaks. However, I suddenly have two new problems, which somehow must have to be related to this RNase treatment: First, in the FL2-W/FL2-A plot, I observe two long, horizontal lines for the G1/G0 and G2/M populations (indicative of massive cell clustering?). Second, additional smaller peaks appear in the FL2-A histogram, as if there were cells containing 6n and 8n of chromosomes. The latter problem, I haven't found anywhere in literature. I have attached an example of the FL2-W/FL2-A plot.

These are the protocols, which I have used:

Without RNase treatment:
1) Trypsinise cells, wash them once in citrate buffer and fix them by adding 100% EtOH forcibly expelled form the pipet. Vortex shortly and store at -20C (at least o/n)
2) Centrifugation, resuspension in citrate buffer
3) Addition of PI / Na2HPO4 buffer immediately before FACS analysis

With RNase treatment:
1) Heat inactivation of contaminating DNases in RNase solution (99C, 5min) and storage in aliquots at -20C
2) Steps 1+2 as described above
3) Addition RNase + PI / Na2HPO4 buffer 37C, 30min
4) FACS analysis

Does anyone have a clue, what these problems could mean and why they appear only after prolonged incubation with the PI buffer or RNase?

Thx in advance for you hints!
Attached Image

-Bonsai-

You have to check the distribution of your cells on FL2-A vs FL-2H dotplot to exclude aggreagates from the analysis.

-Denis Baev-

Hi Denis, I am using FL2-W vs. FL2-A to exclude aggregates because I find this most frequently described. But FL2-H vs. FL2-A would certainly also work for this purpose. I am pretty sure that the long "tails" would also turn up in that plot, and the additional peaks in FL2-A anyway.

-Bonsai-

Can you decrease your FL-2 voltage? That may help to get rid of the horizontal lines. I have also repeatedly observed the additional peaks (also using RNase). If you follow www.unmc.edu/media/cellanalysis/cell_cycle_fundamentals.doc you'll find a solution: simply gating them out. Which I find close to cheating, but hey, it looks good, so who cares? :P

-Rsm-

Very interesting, PostDog! So you also observe (weaker) horizontal lines/additional peaks beyond the G2/M peak?! Did you ever find this problem mentioned somewhere?

Concerning the FL2 voltage: I adjusted it in such a way that the G1 peak is at 200 in FL2-A and G2/M is at 400. This is also what is recommended by the manual that you mention. I would have no problem to gate the debris out, if there was a clearly distinct single cell population but you just cannot tell, where the debris starts! The manual you cite says the same (" When analyzing the data in the cell cycle analysis program, use the single cell gate to exclude debris and aggregates -- so long as they are not continuous with the single cell population.")

-Bonsai-

Well, I would say that the manual that I mentioned shows them as well in every scatter plot... Not as pronounced as your data, but quite similar. I would gate this data out, you never know if your cells are maybe 8n, and what impact that may have on their cell cycle. Here's some more data: http://science.cancerresearchuk.org/sci/facs/facs_major_apps/cell_cycle_analysis/propidium_iodide/?version=1.

-Rsm-

Thx for the interesting link, Rsm!
There is one fundamental difference: The horizontal lines in this manual and the one you mentioned earlier only show doublets ("horizontal lines") behind the G2/M population, whereas I observe also a line behind the G1/G0 population (+ additional lines at higher "n"). If I assume the line behind the G1/G0 population (2n) are doublets, the single cells would be 1n and that is certainly not the case! So I think gating them out is not allowed here.

-Bonsai-