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Protocol for flow cytometry on mononuclear cells from mouse blood - (Mar/26/2014 )

Hallo all.
I´m new to flow cytometry and are setting up a protocol for flow cytometry on mononuclear cells from mouse blood, but would like some input from people with experience.
I have bought a kit from Cedarlane (Lympholyte®-Mammal Cell Separation Media (Isolation of lymphocytes from Mammalian peripheral blood), sterile liquid) to isolate the mononuclear cells. But I still need some input like: 
How much blood is needed to get how much separated cells ?? How many mice are needed ?? How do I collect the blood ?? 
I´m in need of a protocol for collection of blood and staining of isolated cells.


How common/frequent are the cells you're looking for?


For relatively common populations (mouse leukocytes are predominantly mononuclear), we've used as little as 50 uL.


For very rare populations (say, dendritic cell subsets, or tetramer-positive cells), you may need to essentially exsanguinate the animal via cardiac puncture.