I am setting up an antigen proliferation assay using Monocytes Derived Dendritic pulsed with KLH in co-culture with lymphocytes isolated by elutriation technique. I perform a CD4 negative selection using MACS beads, then I stain with CSFE or CPD and co-culture them with DCs.
The problem is that after CFSE or CPD staining I lose 50%-60% sometimes even 80% of the CD4 T-cell. I am staining 20 M CD 4 T-cell with 5 uM CFSE or CPD, does anybody have the same problem?