I use 7AAD and ANNEXIN-V-FITC to detect cell apoptosis on a flow cytometer. I have some questions I would like to ask you. Regarding the FSC and SSC gates, should the group of cells on the upper left be circled? I have tried not to circle and to circle, and the resulting graphs are very different. The main thing is that if I circle, how can there be a positive group in the blank tube without dye added? Is it because the dead cells or debris have stronger autofluorescence? If I draw a cross gate according to the position where the blank tube is double negative, there is obviously a problem in the grouping of the fully stained specimens. Below are my analysis graphs with and without circles.
The problem is that you have multiple populations, and each one has its own flourescence. what is your sample? Did you discriminate the doublets first?
Some cells have a higher autofluorescense when they die.
I totally agree with Jahaziel. or you can seat with the FACS team, and they can help you to setting your experiments. Good Luck.