Fig. 5. LPS but not caffeine induces nuclear translocation of NF-kB, whereas caffeine-induced maturation is sensitive to cyclosporine A (CsA). iDCs were left untreated or stimulated for 5 or 60 minutes at 37°C, as indicated. Cells were allowed to stick to poly-L-lysine-treated coverslips and permeabilized with acetone:methanol. Cells were incubated with rabbit anti-p65 polyclonal antibodies (Ab), followed by Alexa Fluor-488-labeled secondary Ab and visualized with a confocal microscope as indicated in the Materials and Methods section. Arrowheads indicate nuclear fluorescence. Images were acquired with a 100x Plan Neofluar oil immersion objective (NA 1.3) mounted on a Zeiss Axiovert 100 confocal microscope. Horizontal bar, 10 µm. Arrowheads point to nuclear translocation of p65. (B) Surface expression of CD83 in unstimulated iDCs (empty bars) or iDCs stimulated with 1 ng/ml LPS (dark-grey bars) ± 10 mM caffeine (light-grey bars) or pretreated with 2 µM CsA for 30 minutes and then with 10 mM caffeine + 1 ng/ml LPS (slashed grey bars), or pretreated with 10 µM deltamethrin for 30 minutes and then with 10 mM caffeine + 1 ng/ml LPS (hatched grey bars). Results are expressed as percent (mean ± s.e.m.; n=3) induction of CD83 surface expression: 100% was the value obtained by stimulating cells with 1 µg/ml LPS for 4 hours. ^**P, significant difference in the treated population compared with iDCs treated with 1 ng/ml.