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Need guidance on FACS analysis - (Oct/13/2008 )

I wonder if anyone can point me in the right direction. I'm just starting 'hands-on' flow cytometry and am looking for a simple guide (of the 'increase FS and your population will move in X direction' type).
I've done all the theory, but now I need to put it into practice, it's a bit scary and a bit confusing! I've also seen the Invitrogen guides posted by Minnie Mouse (thanks - they were great), but again they mainly concentrate on how the instrument works.
Sorry if I seem a complete idiot, but I'm feeling a bit lost at present.

-flow newbie-

Please tell us what analysis you are doing? DNA content, sorting, surface marker?



I would like to start with cell surface markers on PBMC, then move on to intracellular cytokines, if I get the hang of the surface staining.

I've been kind of left on my own as my line manager left quite suddenly, and he was the only one who knew about flow (v. small organisation!). I've read around quite a lot, and have been to a few talks, but got a bit flustered when I actually sat down in front of the instrument. The other thing I find a bit difficult is that our instrument has a plate loader, so I only have a small volume to play with, before it finishes aquiring each well.

The project I need to take over will be looking at CD markers on PBMC of normal healthy volunteers. If I get that set up I would like to move on to stimulating the cells with mitogen and various other compounds, then looking to see how cytokine expression is changed, compared to mitogen alone. I really want this to succeed, and appreciate any help given. Thanks.

-flow newbie-