Figure 2. T. denticola adherence to and uptake by PDL cells.

Panels A and B: T. denticola at MOI=100 was added to PDL cultures for 2h, after which PDL cells were treated (“uptake”) or not treated (“adherence + uptake”) with 200 μg ml^-1 gentamicin for 1h to kill extracellular bacteria. After washing and incubation in fresh αMEM for the indicated times, PDL cells were lysed with sterile water, and lysates were mixed with NOS semisolid medium and incubated anaerobically at 37°C. Panel A: T. denticola colony forming units recovered per well of PDL cells (approximately 10⁵ cells) after 0, 7 and 24 h post-challenge incubation. The data represent two independent experiments conducted in triplicate. Panel B: T. denticola colonies recovered from PDL cell lysates in a representative experiment. Panel C: Immunofluorescence microscopy of PDL cells with or without 2h T. denticola challenge (2h, MOI=100) followed by washes, with or without further incubation in culture medium and membrane permeabilization. Slides were probed with rabbit anti-T. denticola Msp IgG followed by Alexa 555-conjugated goat anti-rabbit IgG to detect T. denticola and phalloidin-647 to detect cytoskeletal actin in PDL cells. Panel D: Imunofluorescence microscopy of PDL cells challenged with T. denticola (2h, MOI=100) followed by washes and membrane permeabilization, probed with rabbit anti-T. denticola whole cell IgG and mouse anti-LAMP1 IgG followed by fluor-conjugated secondary antibodies.