Fig. 3.

Influence of A549 cell coculture on the mobilization of [Ca²⁺]_i and LO translocation in MDM. MDM were cocultured with A549 cells or kept in monocultures in a Boyden chamber (see Fig. 1a) for 48 h in the presence of IL-4 and separated from A549 cells. a Measurement of [Ca²⁺]_i in Fura-2-AM-loaded MDM resuspended in HEPES-BSA buffer containing 1 mM Ca²⁺ and exposed to E. coli (O6:K2:H1; ratio = 1:50) or PBS (vehicle) at 37 °C for up to 90 min. The ratio of absorbance at 340 vs. 380 nm reflecting [Ca²⁺]_i of MDM stimulated with E. coli versus unstimulated cells over 90 min (left panel). Data are given as AUC of MDM stimulated with E. coli subtracted by the AUC of unstimulated MDM (right panel), means ± S.E.M., n = 3 separate donors. Two-tailed t test. b MDM were challenged with E. coli (O6:K2:H1; ratio = 1:50) or PBS (vehicle) for 90 min at 37 °C. MDM on cover slips were fixed, permeabilized, stained with antibodies against 5-LO (red), FLAP (green), and 15-LO-1 (cyan-blue), and then analyzed by immunofluorescence microscopy. Scale bars = 10 µm (upper panel), 20 µm (lower panel). Results shown for single cells are representative for approx. 100 individual cells analyzed in n = 3 independent experiments