Figure 2. Annexin V staining of explant culture cells.

Cultures were incubated with 197N B. avium (10⁸ cells ml⁻¹, Fig. 2a, b), EBSS (Fig. 2c, d), B. avium LPS (Fig. 2e, f), 10⁻¹ dilution of a cell free supernatent (Fig. 2g, h) or B. pertussis Tohama 1 (10⁸ cells ml⁻¹, Fig. 2i, j), for 6 h and stained with annexin V Alexa Fluor 488. Fig. 2a, c, e, g, & i are DIC images of the same field as the fluorescence images of the annexin V staining (Fig. 2b, d, f, h, & j). In these examples 10–20 s video sequences of the movement of the cilia were also recorded, confirming that the fluorescence was associated with the cilia (videos 3, 4, & 5). Comparing the fluorescence image (Fig. 2b) with the DIC image (Fig. 2a) and video sequence (videos 3 & 4) showed all of the ciliated cells in cultures treated with 197n had positively stained with annexin V. Areas where there were no ciliated cells had no discernable fluorescence, demonstrating that non-ciliated epithelial cells did not have an immediate annexin response to the presence of bacteria. In the EBSS treated cultures the cells that showed positive staining (white circles, Fig. 2d) were round, non-ciliated cells in the DIC image (black circles, Fig. 2c). Actively beating ciliated cells (black boxes, Fig. 2c, video 5) showed no annexin staining (white boxes, Fig. 2d). B. avium LPS also failed to induce annexin staining of ciliated cells, as again the active cilia (black boxes, Fig. 3e) showed no fluorescence (white boxes, Fig. 3f). A 1/10 dilution of bacteria free media did induce annexin staining (Fig. 2h, white boxes) of active ciliated cells (Fig. 3g, boxes). Treatment with B. pertussis also did not cause an annexin response (Fig. 2j, black boxes) in active ciliated cells (Fig. 2i, boxes). Scale bars= 50 µm.