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. 2022 Jan 26;11(2):159. doi: 10.3390/pathogens11020159

Figure 6.

Figure 6

Osteoclast differentiation of THP-1 cells with and without LtxA. All cell cultures were differentiated with RANKL and α-tocopherol from day 4–9. LPS and/or LtxA were added as stimuli on days 1–3 and/or days 4–9. Control cells were differentiated with PMA (day 1–3) and with RANKL and α-tocopherol (day 4–9) (A) Microscopy images of cells differentiated under different conditions. Green: nuclei stained by methyl green, red: tartrate-resistant acid phosphatase (TRAP). White arrows: multinucleated cells. (B) The size of osteoclast-like cells and (C) number of nuclei seen per cell. LPS + LtxA: cells were incubated with both stimuli on days 1–3, followed by incubation with LtxA on days 4–9 in addition to RANKL and α-tocopherol. Kruskal–Wallis test, *** p ≤ 0.0001 and * p ≤ 0.05 compared to control, ## p ≤ 0.01 compared to LPS (medians and ranges, n = 2 independent experiments in duplicate). (D) Pit formation ability of osteoclast differentiated THP-1 cells on 40 µm thick bovine cortical bone slices. Differentiation was achieved by exposing cells to PMA for 3d, followed by stimulation with RANKL, α-tocopherol with or without LtxA. Light microscopy was performed, and the surface area of pits in percent was determined by color filtration and binarisation. Two-tailed unpaired Student’s t-test, p = 0.23. (mean ± SD, n = 2 independent experiments in duplicate).