We are dealing now with a problem of auto-fluorescence. Our samples (feval sample of chicken) exhibit fluorescence in green and red channel without adding probes.
The answer depends on whether you want to exclude the autofluorescent cells, or whether your cells of interest *are* autofluorescent. Excluding them is easy: simply leave one channel free as Berislav suggested for the autofluorecent population.
However, it sounds as though you want to analyse cells that are naturally autofluorescent. This is harder. You have two potential approaches: The first is to use fluorophores that emit in channels that are not known for autofluorescence. Broadly speaking, the redder you get, the better. For example, APC and APC-Cy7 are better than FITC and PerCP. The other (not mutually-exclusive) option is to use very bright fluorophores in the channels that do exhibit autofluorescence. This means using Alexa488 instead of FITC, PerCP-Cy5.5 instead of PerCP, and favouring biotin-streptavidin and primary-secondary steps to bolster signal. PE and APC are generally good fluorochromes, and the Brilliant Violet dyes are pretty strong as well. FITC is weak.
Please note that autofluorescence cannot be compensated out of a channel of interest. It is a real signal, and as such "belongs" in the gate, as annoying as it may be. Attempts to quench the autofluorescence will likely do more harm to your cells than good, and are unlikely to be successful in any case. The best approach is not to try to remove the autofluorescence but rather to get your signal sufficiently bright that it no-longer matters.
Are you trying to analyze any stained marker in your samples? It may be possible that some cells give autofluorescence during flow cytometry but this shouldn't be a problem as the autofluorescence signal picked up in the stained sample can be eliminated by the unstained control.
We use unstained control, but also this, give as a fluorescence signal in green, but more in red channel. To be clear autofluorescence increases rapidly by ading hybridisation buffer.
Dear Eva, every cell has some auto fluorescence... So, Flow cytometer will detect all type of cells, and the thing is based on size and morphology, you have to "gate" your cells of interest and they should be labelled with some dyes (eg: PE, FITC, APC etc.....). So that your instrument will consider only those cells and you can be analyse it. I think I am clear to you, if not pls excuse.
I would suggest you add one dye (channel) to your staining for auto-fluorescent cells and leave that channel free of antibodies. In example: you want to stain cells for markers with antibodies labeled with FITC and PE. While acquiring the cells you also acquire the signals in one additional channel (let's say PerCP), you can use PerCP channel to exclude auto fluorescent cells from the analysis. In this case, just be sure to make proper composition, so in this example compensation for 3 (FITC, PE and PerCP) and not 2 dyes (PE and FITC). I find it very useful in my work. Of course, it depends whether you can add one more dye to your staining panel.
I have worked with cells with high natural auto-fluorescence - if these are the cells you actually need I would suggest either avoiding those channels when choosing fluorchromes (tough, I know) or making sure that the signal from your fluorochromes is significantly higher than your autofluorescence (it's not perfect but you can check the specificity of the signal by fluorescence microscopy)
Eva, all though i would not recommend this approach normally as you loose some sensitivity in your population descrimination, you could try reducing your voltages slightly on the channels with the high autofluorescence. As long as you have a significant difference between your positive and negatively stained cells you should still be able to analyse your samples.
I have also heard of methods involving the use of stains such as trypan blue post fix and perm on cells to reduce FITC autofluorescence in hybridisation experiments.
Thank you Anthony, I already did, you adviced me. I will think about to use trypan blue.
The answer depends on whether you want to exclude the autofluorescent cells, or whether your cells of interest *are* autofluorescent. Excluding them is easy: simply leave one channel free as Berislav suggested for the autofluorecent population.
However, it sounds as though you want to analyse cells that are naturally autofluorescent. This is harder. You have two potential approaches: The first is to use fluorophores that emit in channels that are not known for autofluorescence. Broadly speaking, the redder you get, the better. For example, APC and APC-Cy7 are better than FITC and PerCP. The other (not mutually-exclusive) option is to use very bright fluorophores in the channels that do exhibit autofluorescence. This means using Alexa488 instead of FITC, PerCP-Cy5.5 instead of PerCP, and favouring biotin-streptavidin and primary-secondary steps to bolster signal. PE and APC are generally good fluorochromes, and the Brilliant Violet dyes are pretty strong as well. FITC is weak.
Please note that autofluorescence cannot be compensated out of a channel of interest. It is a real signal, and as such "belongs" in the gate, as annoying as it may be. Attempts to quench the autofluorescence will likely do more harm to your cells than good, and are unlikely to be successful in any case. The best approach is not to try to remove the autofluorescence but rather to get your signal sufficiently bright that it no-longer matters.
i'm interested if trypan blue worked for you? and if yes, at what concentration?
I have the same problem....
thanks
Hello Eva, Several groups have utilised trypan blue to quench autofluorescent emission. This publication maybe of interest to you.Article Trypan Blue staining method for quenching the autofluorescen...
Best wishes
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