I am doing a time course experiment to study cell divisions. I have carried out the experiments in dark, given the light sensitivity of CFSE. I have analysed stained and unstained samples at every time point. The samples were fixed and stored for a maximum of 1 week before analysis. I set the voltages based on stained 0 hr samples and acquired others without any change.
Fluorescence histograms seem to have shifted a little between time points. This is apart from the dilution of CFSE due to cell division. For instance, in Fig 1, at 24h I see a single parent population with fluorescence much less than the 0h sample. I know that division would not have occurred, based on cell cycle analysis. This continues at later time points also. After 48h, I cannot make out if a short high-intensity peak is the shifted (bleached?) parent population or diluted from initial cell divisions.
This continues at later time points also. After 48h, I cannot make out if a short high-intensity peak is the shifted (bleached?) parent population or diluted from initial cell divisions. In Fig 2, I feel the peaks marked by red lines are the same division population; similarly with blue lines.
Which tool should I use for analysis and how do I compensate for the shift due to bleaching/quenching during cell growth and handling?
The main problem in your assay is the one week delay in time before the measurements. The dye fading might be negligible (also you might need to use novel anti-fade reagents for flow), but the dye also leaks out of cells during storing in the solution for several days. You wrote that "samples were fixed and stored for a maximum of 1 week before analysis". Cells for flow cytometry are fixed and usually stored in solution at 4 to 8C. You need to analyse your cells next day or even the same day if possible to avoid leakage and transfer of dye from cell to cell. There is new dye developed to prevent leakage:
https://www.sigmaaldrich.com/technical-documents/articles/biology/fluorescent-cell-dyes.html
https://www.sigmaaldrich.com/content/dam/sigma-aldrich/life-science/cell-biology/isac_06_cellvue_claret_poster.pdf
According to your figures it looks like the cell populations are the same, and the shift is most likely due to leakage of dye from cells.
The main problem in your assay is the one week delay in time before the measurements. The dye fading might be negligible (also you might need to use novel anti-fade reagents for flow), but the dye also leaks out of cells during storing in the solution for several days. You wrote that "samples were fixed and stored for a maximum of 1 week before analysis". Cells for flow cytometry are fixed and usually stored in solution at 4 to 8C. You need to analyse your cells next day or even the same day if possible to avoid leakage and transfer of dye from cell to cell. There is new dye developed to prevent leakage:
https://www.sigmaaldrich.com/technical-documents/articles/biology/fluorescent-cell-dyes.html
https://www.sigmaaldrich.com/content/dam/sigma-aldrich/life-science/cell-biology/isac_06_cellvue_claret_poster.pdf
According to your figures it looks like the cell populations are the same, and the shift is most likely due to leakage of dye from cells.
Bleaching due to autooxidation also possible. May be it possible to introduce control cell with blocked mitosis in experiment to have appropriate day-to-day possitive control.
In Fig 1, are you resting your 0 h (parent population) overnight, prior to fixation? CFSE stained cells will efflux the CFSE overnight, reducing CFSE intensity by about one log (e.g. 10^5 to 10^4), independently of cell divison. This resting step would align your 0 and 24 h CFSE peaks in Fig 1.
I also agree with Olga, analyze same day when possible.
Sherman,
In fig.1, I do agree CFSE could be effluxed out during the first day. But during the subsequent days as well I do see a shift in the peaks. I have tried what Olga suggested. It did help, but only to a small extent. I still cannot overlap data from different days. Any suggestions?
Devi, may I ask what the yellow and black curves in Fig 2 represent? Are they both 48 h samples?
Sherman,
The yellow curve is from 48h sample and black is from 72h sample. I was intending to mean, the shift in the 1st generation peak from 72h sample is so much that it overlap's with the 48h sample's 2nd generation. So I am unable to be sure if the shift is because of dilution due to division, or just loss in intensity.
I did check the same unstained sample on different days. After day 0's analysis, I had fixed the cells with 4% PFA and probably hence the autofluorescence increased by day 1. However, after that, the position of that peak was constant.
That's a way to find out. But looking at some more data, it appears to be more likely that the same peaks are shifting. In the attached images, I have connected those populations by lines, which I think are the same generations. Each pair of distributions is from consecutive days.
The shifts are the worst where I fixed all cells and analysed them together on the last day. The second and third images are of live cells, acquired every day. The shifts are less, but still there. I don't know if data presented like this is acceptable or not.
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