cd4 cells activation cytometry problems

[giannisanton]

Hi, everyone!
I will get straight forward to the point. I defrosted human PBMCs and separated CD4 cells using MACS CD4 isolation kit. I performed a flow cytometry with ECD CD4, the same day, and I found a 97% purity of CD4 cells. I left these CD4 cells overnight with RPMI,pen/strep and 15% FBS and the next morning I stimulated them for 5 hours with PMA and Ionomycin. Afterwards, I stained 100.000 of these cells with human CD40L PE and another 100.000 cells were left Unstained. After washing with PBS I performed a flow cytometry. What I noticed was that I got two different populations very close to each other (actually up and down) for both stained and unstained cells. My first question is:
Could one of these populations be dead cells OR could one of these populations be the activated and the other the non activated cells? I don't know which population to choose; should I choose both?
Moreover, on the CD 40L PE (FL2) histogram (log) plot I get a bizarre view:
When I ran the Unstained Cells I had a certain population. Using the same protocol, I ran the CD40L PE stained cells where I got another population which is pretty displaced to the right and has 2 peaks (actually as if there were two different populations). My second question is:
Could this happen because of the staining with CD40L PE? I mean, does this
antibody have any effect on the CD4 cells apart from marking? The staining with CD 40L PE lasted 40 minutes, if this helps. What I believe is that at the CD40L PE stained cells, the subpopulation on the right (approximately 60%) is the activated cells and on the left is the non activated cells.
I have the same results and questions when instead of PMA Ionomycin I stimulate the cells using anti CD3 CD28 beads.

Thanks in advance for your help