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Hello, I am a tyro about doing FACS analysis. I have tried to learn the basic knowledage about FACS, but when doing the real experiments, I`ve encountered some problems. Here I would like to ask something about the FSC-A oscillograph trace.
About my research, I use the BT-20 cell line, and after incubation with my fluorescent proble, I collect the cells and apply them to the FACS Canto apparatus. But no matter how I adjusted the parameters, I got 2 peaks in the FSC-A oscillograph trace. I really got confused why there would be two peaks(one is sharp and slim, and the other is much broader and lower.) in the FSC-A oscillograph trace. Can someone give any suggestion what I can solve this problem? Previously when I analyzed another cell line HT-1080, I didn`t see two peaks for FSC-A, but now I have no idea about how to analyze this cell line in a proper way. As this BT-20 grows much slower, and I also found much more dead cells in the medium during subcultivation.
Looking forward to hearing some helpful explanation!Thank you in advance!!
Last edited by Crystalbin on Thu Feb 05, 2009 10:30 am, edited 1 time in total.
Based on the dot plot it seems that you may have two cell populations; if you carefully look at the region where the most cells are, there is a thin line separating a smaller set of cells on the right hand side of the P1 gate. You coult gate both of these populations separately and see if one gives the "thin" peak on the histogram/oscillograph and the other the "broad" peak. To me it seems that you just have two different-sized cell populations, that's all.
The reason for that could be that a portion of the cells is undergoing mitosis and are in the middle of the cell division, thus appearing as larger cells. Also with certain cells, if some of them get activated they can be seen as a separate population of large cells. This concerns mainly lymphocytes and such, though, and maybe not BT20. It is also possible that there has been a mutation in some of your BT20, and they now grow larger, thus you could have two types of cells in your culture, although very closely similar.
Also, be sure that you gate only the living cells (unless for some reason you want to include the dead ones as well). Do you have any specific marker for BT20 that you can use to verify this? What was the antibody on the fluorochrome (FITC) you used, and how does that look on a graph?
Thank you very much for your detailed replied. Now when I look back to the histogram, I think I noticed the thin line separating two different-sized cell populations, indeed, that could be due to the much slower growth rate enhanced the risks of the change of BT-20 cells,like mutation or cell division just happened before collecting the cells. But if it is the case, what do you recommend to avoid this situation? Should I try some other faster growing cell line,like MCF7?
Yes, I do hope only gate the living cells,but so far I didn`t use any specific marker to verify the BT-20, I only observed the morphology of the BT-20 cells, and checked if they are attached to the plates, as they are adherent cells. Sorry, I don`t know what do you mean: how does that look on a graph?
Last edited by Crystalbin on Thu Feb 05, 2009 10:31 am, edited 1 time in total.
How does that look on a graph = Is everything ok in FITC channel (histogram/dot plot/whatever you use)? I.e. does everything look ok there, or do you get two peaks on FITC as well?
Maybe the main point here is: does it matter that you have two BT20 populations? If you e.g. only want to see if your peptide enters the cells, just include both populations and see on the FITC channel whether the peptide is there or not? :)
If having two populations is a problem, then you could try to arrest the cell cycle before running the samples. If I recall correctly, there are reagents one can use to "hold" all cells in, say, G1 in interphase in the cell cycle. If the populations then look uniform, the dual population was most likely caused by different cell cycle phases (e.g. late mitotic cells that are bigger than normal cells). Alternatively you can keep your cells a few days without growth-promoting substances (if you use any, such as cytokines, interleukines etc.) and let the cells to "calm down".
If you are unsure about dead cells, you may try using 7-Amino-actinomycin D (7-AAD) [trade name BD Via-Probe] to exclude dead cells on FACSCanto.
Of course, simply using some other cells can do the tirck as well.
Thanks a lot for your quick reply and the suggestion on different possibility.
The FITC-A histogram looks quite okey, there is only one sharp peak. So that`s why I wonder why there would be two peaks in FSC-A histogram.
I will take your suggestion to use the 7-ADD for excluding the dead cells in the future experiments.
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