The cells were expressing GFP and also incubated with PI for dead cells detection.
This is my first-time using this BD Accuri C6 and I'm not sure if I need to set compensation (I tried to follow protocol to set compensation but failed to do so) or simply the filter was not working. Thank you very much!
Dear Zhonglei He,
I assume this is caused due to the failed compensation. Since PI as well as GFP show huge spectral overlap in the respective other channel you have to compensate for this.
Perform a single staining of PI (just PI, no other fluorophors) and a "GFP stained" (use cells expressing gfp quite strongly) sample. Than you have to subtract the overlap manually. Look at the single stainings and check the window for compensation and axis labeling. If PI for example causes signals in other than FL1 (if I recall the channel correctly) substract them by setting percentages in the corresponding Matrix (substract X % of FL1 in FL2; for example). Do the same thing for GFP.
Hope this helps
all the best
Robert
This is nothing but compensation issue. You need to substrate FL1from FL2 and FL1 from FL3.
Your GFP signal is offscale. Reduce the GFP voltage setting and the compensation should work.
cheers,
ann
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