I am new to flow cytometry, and was just using a sample cell population to train with the technician. The cells were PAC-1 cells split into four groups. Unstained, transiently transfected with mCherry, DAPI stained (for live/dead fluorescent gating) and a transfected+stained group.

During the process of establishing voltage & compensation levels for the different single-stain populations, there was an abnormally high amount of DAPI-channel fluorescence (majority of cells in the 10^4 and 10^5 area) from the mCherry-only population.

This stumped both myself and the FC tech, as there should be almost no emission overlap between the mCherry fluorophore and the DAPI emission filter. Contamination also seemed unlikely as the DAPI expression was consistently high in all mCherry+ cells, not just dead ones (as it was with DAPI+ only).

If anyone has any guesses as to what may have caused this, I would greatly appreciate it!

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