I have previously compensated with single color controls. For my current flow cytometry work, the lab has adapted to using only FMOs for compensation of the fluorophores. Working with cell types that are very limited (a few hundreds per mouse) in number would make it difficult to have both single color controls and FMOs. However, would just using FMOs for compensation be accurate? If not, what is the best solution for compensation and gating?
Hi Nikhil,
it is definitely not recommended to run any flow experiment completely without single-stained controls. What you are referring to (and what is indeed done by some people) is probably doing just manual compensation based on the visual appearance of the FMOs - an approach that can be very problematic.
Since you were mentioning your low cell numbers - this should not be a limiting factor, as for your single stained controls you do not have to use cells, but you can (and should) use antibody capture beads. If you want, you can read more about this topic here:
Article Guidelines for the use of flow cytometry and cell sorting in...
Chapter Basic Multicolor Flow Cytometry
Good luck with your future experiments!
Thank you for your replies. I really appreciate the input. Gulab Fatima Rani
The problem with using cells for these extra controls, is that I am trying to do a quantitative analysis of neural stem cells from single mice and I am already limited with cells which in the current setup are in the range of 150-200 cells in total. Then within these I have specific sub populations that I am assessing. I resuspend my cell pellet in 120ul FACS buffer and use 20 ul for the FMOs. Already, the to gate certain markers I have 10-20 events only which is already not very effective to determine the range of fluorescence. So using additional cells for single color controls is not a feasible option. But, like Florian Mair suggested may be the beads for the compensation would work for me in this scenario. Thank you once again.
Use antibody labelled beads to set compensation. FMOs should not be used for this purpose since often the quantity of rare cells will be too low to set accurate compensation. Although most people define FMOs as gating controls only - they can be used to see if the compensation is accurate, i.e if for example a single colour bead control has an antibody of less intensity than the actual sample, the compensation will be inaccurate and the FMO will enable you to see this as the overlap into other channels will be evident.
cheers
Ann
How many colors are you using for your FACS analysis?
If you are using 3 to 4 colors, try to choose colors which are not overlapping and shows peak at very far in spectrum when exited by laser. In this case you don’t need to compensate repeatedly and you can fix gating strategy by using FMOs. If the number of colors are too many then you need to use extra mice except from your experimental group just for compensations.
Hope this can help
All the very best
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