The Influence of Ouabain on Human Dendritic Cells Maturation

Figure 8

Evaluation of plasma membrane potential in the presence of Ouabain. (a) Differentiated DCs (maintained 5 days with GM-CSF and IL-4) were further incubated in the presence or absence of 100 nM Ouabain or 1 mM Ouabain with 250 nM of the fluorescent oxonol dye DIBAC₄(3) for 30 min. (b) Differentiated DCs were incubated with or without TNF-α in the presence or absence of 100 nM Ouabain for further 48 h. Then, cells were washed, free medium was added, and DCs were incubated with 250 nM DIBAC₄(3) for 30 min. As a control of membrane depolarization, 50 mM KCl was added for 30 min (during incubation with DIBAC₄(3)) to the cultures. Next, cells were analysed using flow cytometry, as indicated in Material and Methods section. Data depicts a representative experiment from one individual ((a), 5 independent experiments; (b), 2 independent experiments) and values in each panel refer to the means of fluorescence intensities (MFIs) of DIBAC₄(3).

(a)

(b)