Hi Dear all scientists, I am a beginner in flow cytometry. Recently I did cell cycle experiment with myeloma cell line using BD FACSVerse system installed BD FACSsuite software. I made 2D dot figure and histogram figure I attached. But I found out that positions in G1/G0, S and G2/M often moved in graph. Especially in histogram figure it shifts in x-axis. I don't know what it causes. Thank you so much.
Are there also fluorescence signals in the other channels (form antibodies, EdU,...)? Maybe it's a problem of your compensation: in one sample there might be a strong signal in another channel, which is than subtracted from your 7-AAD signal. In another sample, there might be a lower signal which is subtracted from your 7AAD signal, and therefore it shifts on X-Axis in histogram.
Hi Shiqiao,
This usually happens if the cell number between samples are considerably different. You can count the cells before staining with PI to ensure you have same cell density, otherwise, you could adjust the settings before running each sample so that 2n peak is aligned between samples.
Regarding the figure, normally x-axis should be labelled PI unless you permeabilised the cells and used 7-AAD.
I guess you used the BrdU or EdU Kit. Most common reason is simply the concentration of your 7AAD staining is slightly different. The amount of buffer or even time differences till you actually facs can have those effects. I found that it is simpler and even more price efficient to use DAPI.
If your FACSVerse has a UV or violet laser, just mix it in the buffer you use to resuspend your cells, wait 5 minutes and facs. This way you will have always the same results, even if you can’t use the machine right after your BrdU or EdU staining. It’s also working with PI if you don’t have the laser.
Thanks for all scientists. Yes, I use BrdU kit from BD. Next I will count cell number and keep the same. We have a Violet laser. So I will also try DAPI. Maybe after 2 weeks, I will feedback my results to all.
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