Invertoskop 40c microscope

OECs were seeded in 12-well plates and infected with an MOI 100 of either P. gingivalis 33277 or F. nucleatum 25586 for 72 h. Cell culture media was changed at 24 h intervals. After 72 h of infection a scratch was made in each well using a pipette tip. Cells were monitored for migration in fresh media containing the proliferation inhibitor, Mitomycin C (5 μg/mL). Migrating cells were monitored at 12 h using Zeiss Invertoskop 40C Microscope. The images were captured using a cooled charge-coupled device camera controlled by QCAPTURE software (Qimaging).

CFU assays were performed with MethoCult GF (M3434, Stem Cell Technologies) according to the manufacturer's instructions as described previously [8] (link), [33] . BM-mononuclear cells (BM-MNCs; 2×10⁴) were plated in triplicate in 12-well plates and were incubated at 37°C in 5% CO₂; colonies of burst-forming-unit–erythroid (BFU-E) and CFU–granulocyte macrophage (GM) were scored on day 7; those of CFU-granulocyte, -erythrocyte, -monocyte, and -megakaryocyte (GEMM) were scored on day 12 of the incubation. In both sets of plates, colonies consisting of ≥40 cells were enumerated by using a Carl Zeiss Invertoskop 40C microscope with a 10× objective. Colony counts were expressed as plating efficiency, which is the number of colonies per 2×10⁴ nucleated BMCs.

A sterile glass cylinder (Bioptech) was placed in the center of each well in a 6-well plate. OECs were seeded in the 6-well plates outside of the glass cylinder thus creating a Zone of Exclusion (Nyegaard et al., 2016 (link)). The OECs were then infected with an MOI 100 of either P. gingivalis 33277 and/or F. nucleatum 25586 for 72 h. The glass cylinder was then removed and cells were monitored at 12 h for migration in fresh media containing the proliferation inhibitor, Mitomycin C (5 μg/mL) using Zeiss Invertoskop 40C Microscope. The images were captured using a cooled charge-coupled device camera controlled by QCAPTURE software (Qimaging).