I've been using MoFlo xdp to sort bone marrow samples to isolate plasma cells... When I exclude the doublets there is always one big singlet population... But last time I got two big populations on the FSC-W.
No, it's not normal. You say it only happened last time: have you done anything that could make the cells stick together? I know that various fixation procedures can make cells sticky, but this seems quite extreme.
I use BD software for FACS, so I can't relate to the exact placement of the populations on the axes in your figure, but you should at least make sure that your signal width of the doublet population is ~ 2 times the width of the singlet population.
If this is not so, I would suspect that your singlet population is actually noise of some sort, and that you doublets are actually the singlets.
If no explanation seems valid, try repeating your experiement and see if the problem persists.
Paola, it would be very helpful if you show your previous experiments (those that you consider normal), also, could you please include FSC vs SSC plots? (or simply attach fcs files).
Paola, thank you for sharing the file. Your "normal" file looks very clean. It seems that your current preparation indeed has a lot of doublets (probably due to cell death/damage during sample preparation, which leads to release of genomic DNA and cell aggregates formation). Pay attention to sample preparation and make sure to filter it before sorting. Good luck.
Hi Paola,
it´s interesting that you normally just get one single population and do not have cell aggregates; I unfortunately couldnt open the files, but I like the answers from Thomas Haupt and Alexander Misharin.
One more explanation: do you normally have more cells (where you see just one population)? Then it could be also possible that the both population melting together in the density plot... depending on the choosen resolution... just an idea.
I have the same problem with my MoFlo-XDP. When I chanche the hight of the FS than the 2 populations goes together to one nice population.
No one can give me an explanation for this phenomenon.
Now i have 3 Population only with FS-W. But SS-W vs SS-H looks normal (1 population and 5% dublettes).
I phoned with Mr. Egner (application specialist from Beckman/Germany) and he told me that I dont have to use the Parameter FS-Width by MoFlo machines to discriminate doublets, because FS-W is realy not the time-of-flight but only the half of FS-H. It is not the same like BD machines. So I had to use FS-A vs FS-H and discriminate all events which are not located on the diagonal. So I am sorting my schwann cells with both plots SS-W vs SS-H and FS-A vs FS-H and I am discriminate 10% doubletes and the purity of the sorting was 99%. So I think i will prefer this way of discriminating doubletes when sorting with my MoFlo.
If you change the Forward-scatte hight your 2 populations change to one population and every looks normal. But I will use in the future FS-A vs FS-H and SS-W vs SS-H to discriminate dublettes.
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