I had a question for the Flow experts here. Has anyone tried staining with multiple dyes - 7-AAD, Annexin-V (PE) - part of Annexin V Apoptosis Detection Kit from BD Pharmingen. These are to be coupled with Dyecycle Violet (BV421) and Cell Trace CFSE (FITC). I have the following problems -
1) 7-AAD seems to be bleeding into the PE channel. I've tried compensating and I have been unable to do so
2) Also Annexin V (PE) is bleeding into the Dyecycle channel.
3) Cell Trace is bleeding into Annexin V and 7-AAD channels. I've tried knocking them down.
I have already tried compensation. Automatic and manual. I am running my samples on the FACS-Canto-II.
Has anybody else tried this combination or has experienced similar issues?