The results obtained with primary human blood monocytes

could be confirmed by the use of the two cell lines. As shown in Table 1 RAW264.7 infected with BCG (pAS-MDP1) had formed 5.1 times more multi-nucleated cells after five days than RAW264.7 infected with BCG (pMV261). The cell line MM6 presented 3.2 times more multi-nucleated cells after infection with BCG (pAS-MDP1) than after infection with the reference strain Blebbistatin mw three days after infection (Table 1). The different cell types varied with respect to maximal fusion indexes reached. Upon infection with BCG (pAS-MDP1), for example, RAW264.7 achieved the highest fusion index with 27.2% followed by human blood monocytes with 15.1%. The lowest fusion activity was observed with MM6 cells that only reached a fusion index of 7.4% (Table 1). The different types of monocytes furthermore differed with respect to the morphology of the fused cells (Figure 5). The morphology typical of Langhans cells characterised by nuclei arranged in a circle along of the periphery of the cell was only present in human blood monocytes (Figure 5A). RAW264.7 cells were shaped more irregularly, and the nuclei were concentrated in the central part of the cells (Figure 5C). Multi-nucleated MM6 cells were strongly enlarged, round, and the nuclei were spread relatively evenly across the cells (Figure 5B). Figure 5 Morphology of multi-nucleated cells. Human blood monocytes (A), MM6 cells (B)

and RAW264.7 cells (C) were infected with BCG (pAS-MDP1) and stained with Diff-Quick. Batimastat cell line Micrographs were taken with a magnification of 400 × . The fusion process then was analysed in-depth AG-120 purchase by calculating the fusion indexes with respect to the number of nuclei per cell.

Figure 6 is a graphic illustration of the distribution of the fusion indexes in the cell line RAW264.7. The uninfected cells generated multi-nucleated cells up Carnitine palmitoyltransferase II to only seven nuclei per cell. Up to eight nuclei per fused cell were present in RAW264.7 infected with BCG (pMV261). Much more fused cells with much higher numbers of nuclei were present in the LPS/IFN-γ-activated cells as well as in cells infected with BCG (pAS-MDP1). The highest number of nuclei per cell was found in cells infected with BCG (pAS-MDP1) with 13 nuclei per fused macrophage. From this illustration it is obvious that the fusion rates of strain BCG (pMV261) were more similar to those of uninfected cells, while the fusion rates of strain BCG (pAS-MDP1) resembled more those of cells activated with LPS and IFN-γ. Figure 6 Number of nuclei in multi-nucleated RAW264.7 cells. RAW264.7 cells were infected with BCG (pMV261) and BCG (pAS-MDP1) at an MOI 50. Uninfected cells served as negative controls and cells activated with LPS and IFN-γ served as positive controls. Five days after infection the cells were stained with Diff-Quick, and the nuclei per multi-nucleated cells were counted and the fusion indexes calculated.