multicolor panel problems

I am trying to set up a 9 color panel on the FACsAria.	My question
pertains to the staining of CCR7.  Currently, we are trying to do a
three step stain for this receptor using

	    1.	pure CCR7-ms IgM

	    2. anti ms IgM-biotin

	    3. streptavidin-Quantum Dot 525 (emission is close to Alexa
430)

 

When I run this three step CCR7 along with anti CD3-PE-Cy7 and anti
CD8-Alexa 405, I get separation of CCR7 pos and neg populations but upon
histogram viewing, the peaks are almost side by side and not very well
separated.  There's about 0.5 log difference max between the peaks.
However, when I run a 3 step CCR7 with the last streptavidin step being
conjugated to PE, there is good separation between the pos and neg
populations with at least 1.5 log separation.  

 

Since I'm running a 9 color panel, I am a little restricted to which
colors I can use.  That is why I have resorted to using the Quantum
Dots.  Do you think that using a quantum dot that emits in the far-red
region would improve the staining?  I'm thinking about using a
strepavidin-Quantum Dot 800.  However, I still do not understand why
there is such a drastic difference in the staining between the Quantum
Dot 525 and the PE.  

 

Any help would be appreciated.

 

Tina