If I've got a compensation control that is much brighter than my experimental sample, is the compensation value derived from this control still valid? Intuitively, I feel that more fluorescence intensity should equal more spillover signal relative to the dimmer sample. However, most protocols I've come across recommend that the compensation controls used should be as bright as experimental or brighter.
More detail:
My experimental fluorophore is an EYFP fusion expressed as part of a bicistronic gene. The gene was stably incorporated by lentivirus. My compensation control is unfused EYFP expressed from a transiently expressed plasmid. The combination of fusion, bicistronic expression, and relatively lower copy number accounts for the difference in brightness.
Yes compensation controls used should be as bright as experimental or brighter. Your aquisition should be on the same gain of fluorophore for compensation control and experiment samples. After compensating its the increase in events of YFP which will be enumerated based on stably incorporated gene. Higher gain of compensation control will always be there unfused EYFP expressed from a transiently expressed plasmid compared to low copy no.
Try rading your control cells earlier, at 5-6 hours post.trasfection expression should be lower. Not as elegant as one would like, but may be still effective. Minor differences in brightness shouldn't be too dramatic.
The "or brighter" part of the compensation rule points you to the fact your control should be valid for your needs. The compensation algorithm will "scale down" the spillover calculation for each event that is less bright than the control. However, if the brightness of either your experimental sample or your comp control is out of the linear range of detection (too bright control, too dim experimental), your measurements and your compensation would be impaired. Make sure both of those measurements fit into the range.
When I set a compensation I do it in several steps. First I always start out with cells with quite a lot of fluorescense to get a ruff sense of the compensation needed (with single-labled cells, one sample for each antibody/dye). After that I fine-tune the compensation by running the cells with the fluorophore of interest in the concentration I want to use (again with single-labelled cells). And then finally I check everything by running samples with all fluorophores except one (in the intended concentrations), this is called FMO (fluorescence minus one). It takes a little bit of time to set it up, but then again everything else in trying out new antibodies/dyes and creating new protocols takes a bit of time, and once your done you just have to check it from time to time and that goes quickly. Good luck!
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