Fig. 9.

Barrier-body formation and uptake by neutrophils in primary pBCEC cultures. Primary pBCECs were treated with either DOXO (10 µM, 30 min) or EFIG-AM (30 min) after culturing on collagen-coated glass coverslips for 5 d. Depending on the experiment, DOXO- or EFIG-treated cells were incubated with freshly isolated porcine neutrophils. Barrier-body formation and uptake by neutrophils were analyzed. (A) LysoTracker- and DOXO-treated pBCECs were incubated with eFluor670-labeled porcine neutrophils (white) for 20 min at 37 °C. Cells were fixed with aceton-methanol and indirectly stained for Pgp (Materials and Methods). Samples were analyzed by confocal fluorescence microscopy. Lysosomal sequestration of DOXO (1) and neutrophils exhibiting green (Pgp), red (DOXO), and blue (LysoTracker) fluorescence (2) indicate formation and phagocytosis of barrier bodies also in pBCECs. (Scale bar: 50 µm.) (B) Confocal fluorescence micrograph of barrier-body formation (boxed magnification) in DOXO-treated pBCECs. After DOXO treatment, cells were incubated with neutrophils for 5 min followed, by aceton-methanol fixation and indirect staining for Pgp (green). Nuclei were counterstained with DAPI (blue). (C) Similar to DOXO-treated pBCECs, barrier-body formation and uptake by porcine neutrophils (eFluor670-labeled, white) can be observed after treatment of the culture with EFIG (red). Cells were stained with LysoTracker (blue); Pgp (green) was indirectly stained. The overlay of Pgp, EFIG, and eFluor670 (Inset 1) shows colocalization of neutrophils with Pgp and EFIG substrate, as well as LysoTracker (Inset 2), indicating uptake of barrier bodies by neutrophils. Insets in the upper left and lower left show magnification of Pgp-, EFIG-, and LysoTracker-positive barrier bodies at the surface of pBCECs, as well as neutrophils. (D) Scanning electron micrograph of a barrier-body aggregate on the surface of a primary pBCEC after treatment with DOXO.