I am dealing with 3-color panel design now, they are: PE, APC and BV605. If I am gonna use FMO control in experiment, do I still need to do compensation before experiment?
You should not be confused with FMO controls and compensation controls, They serve entirely different functions in an experiment.
You should be using single color stains for setting the compensation. FMO is usually used to check the overflow in some multi colors staining, even FMO is nowadays replacing Isotype controls. FMO helps in designing gating strategy.
As far as compensation is concerned, it can be warded off if you are selecting different channels and colors which are not overlapping. It is always safe to compensate your FACS data to avoid any miss interpretation of results.
I hope it can help
All the very best
Dear Zhengzou,
I just briefly wanted to send you some suggestions for your flow experiment. First of all I totally agree that you absolutely need the compensation controls, as two of your colours are excited by the SAME laser (yellow) and have VERY SIMILAR emission spectra. Please consider
http://www.bdbiosciences.com/ca/applications/s/spectrumguidepage
for additional information.
Besides, here's a great example outlining the differences between FMO, isotype and unstained control, including gating examples. As a rule of thumb, isoype controls are for staining issues; FMO controls are for gating/ population size issues (as Prashant already outlined) whereas unstained controls show you the autoflourescence of your population. I hope that will clarify your question and help you with the design of your experiment.
Here's the link: http://flowjo.typepad.com/the_daily_dongle/2011/09/fmo-vs-isotype-controls.html
Kind regards,
Michael
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