Flow-Cytometric Immune Function Methodology(15)

Flow-Cytometric Immune Function 55

ing 3.2. It is also important to follow the requirement to use polypropylene tubes. DCs can stick to polystyrene material and might be lost from the cell suspension that is analyzed.

4. The staining intensity of the lineage cocktail in activated samples decreases

over the length of the sample incubation time. The longer the blood is activated, the less suf cient is the separation of lin 1 dim versus lin 1 lineage-positive leukocytes—in particular, monocytes (1). Monocytes have a very similar phenotype to CD11c+ DCs and are activated also by LPS, which affects the separation of lin-1-positive events from lin 1 dim DCs (1). Best results are obtained for shortest activation times, ideally 2 h and maximally 4 h. Compare the resolution of HLA-DR vs lin 1 plots for LPS-activated and resting blood in Figs. 3 and 4. TNF-α is the intracellular readout of choice because of its early kinetics over other cytokines (seeFig. 7).

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