Hi Martin,

It's a bit difficult to answer your question without more context. Is the AmCyan channel covering a viability dye/marker? And neither samples (FMO/Unstained) have been stained with this AmCyan-dye? Then I'm guessing that either 1) Your unstained sample has not been compensated, or 2) compensation (or overcompensation) of some other fluorochrome signal in your FMO sample is skewing its population (FMO normally has higher background than the unstained), or 3) you've maybe swapped the tubes. Unless you have a biological control for your marker of interest, your FMO can be used as a negative control to account for signal spillover from other ab's in your panel. I.e. you should use the FMO, and not the unstained, to set the negative gate for your other samples. When it comes to dead/live dyes they should give a clear separation and more often do not require an FMO control.

If AmCyan is not a viability stain, I'd try to include one (PI/amine dye, etc) and a dead population (a positive control), to make sure that the cells you exclude are actually dead!

Best of luck!

This does not seem normal.

It is common to get some background noise in FMO's, but not the other way around. Have you run a positive control for your Amcyan viability marker?

Are you sure the settings have not been changed for the runs? Were these runs back to back?

It looks as if the headings should be swapped.

Lewis Williams I am positive that wells identification were not mistakenly swapped. These runs were indeed back to back, i checked the acquisition settings, there is no change. I agree that this is not normal, and that something went wrong. I was just hoping to understand what could of gone wrong during acquistions/compensations.

Mr. Martin Schnitzler ,

To get a clear picture, we have to start with the

i. Sample preparation - sample must be prepared in the same way

ii. Unstained sample - autofluorescence should be adjusted at the very beginning.

iii. After autofluorescence gating , don't change the PMT voltage.

iv. FMO control should be prepared specifically.

v. Please check Am-Cyan overlapping.

vi. Kindly upload the gating strategy for both unstained and FMO & their duplicate graphs.

vii. Don't forget to use dead cells detecting dye like 7AAD or PI etc.

Best regards,

Babu M.

It depends on the number of fluorescent markers that need compensating. It is not unusual to see a shift downwards in the fluorescence in some channels after compensation. If everything has been set up correctly then the FMO is the correct control for gating.