Memory T-cells

Dear Flowers!

We would like to  look memory cells in vaccinate animals or in human
infected patients. We are interest to look CD62L, CCR7, CD25 T-cells and
production of Intracellular IFN-G and IL-2. If you add CD4, CD8,  CD3
and a tetramer, this begin to make a lot a parameters (8 to 10 colors).
We have presently a LSR II, that have a normal set up of 2 lasers (488nm
and 633nm) and 6 PMT (4 for the blue (octogon), and 2 for the red
(trigon)). We think to upgrade our instrument we more PMT or/and a
violet laser (however a new laser seems to not fit we our budget).

I have some questions.

1-  Are these marker are relevant for the observation of the
memory-cells  (central and effector)? (should we add more, and do we
have to use them all in the same tube)

2-  Beside adding a PMT in the red laser, how many PMTs could we add in
the blue laser (max 3 more), and what combination of fluorochrome would
you suggest us to use?
3-  What are the pitfalls that we could encounter in our process to
develop 8 to 10 colors experiment ? Do you have any goods tips in the
choice of the fluorochrome for the different antibodies?

4- About Conjugaison of anitbodies, does anybody used these Quantum dot
fluorochrome ? How are they? What about the conjugaison method, is it
complicate?

Thanks in advance

Dominic



Dominic Therrien, M.Sc
Research Assistant
Molecular virology dept
Statens Serum Institut
Artillerivej 5
2300 Copenhagen E
Denmark
nic@ssi.dk
45 3268 3579